Nsp molecules

ABSTRACT

The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, antagonists and agonists vectors and host cells expressing, and immunoadhesions and antibodies to PRO201, PRO308 or PRO309 polypeptides. The invention further relates to compositions and method for the diagnosis and treatment of neoplastic cell growth and proliferation in mammals, including humans. The invention is based in part on the identification of genes that are amplified in the genome of tumor cells. Such gene amplification is expected to be associated with the overexpression of the gene product and contribute to tumorigenesis. Accordingly, the proteins encoded by the amplified genes are believed to be useful targents for the diagnosis and/or treatment (including prevention) of certain tumors (e.g., cancer) and may act as predictors of the prognosis of tumor treatment.

PRIORITY INFORMATION

[0001] This is a non-provisional application filed under 37 C.F.R.§1.53(b)(1), claiming priority under 35 U.S.C. §119(e) to provisionalapplication No. 60/082,767 filed Apr. 23, 1998 and provisionalapplication No. 60/113,296 filed Dec. 22, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides which are characterized by the presence of novelSH2-containing proteins (Nsp's).

BACKGROUND OF THE INVENTION

[0003] Interactions between ligands and the cognate cell surfacereceptors are critical for a variety of biological processes includingmaintenance of cellular and organism homeostasis, development, andtumorigenesis. Many of these ligands can activate multiple independentpathways and the strength of the activation of different pathways can bemodulated by the presence or absence of signals generated by otherreceptors, Hotamisligil, et al., Proc. Natl. Acad. Sci. USA 91: 4854-58(1994); Kanety et al., J. Biol. Chem. 270: 23780-84 (1995); Luttrell etal., J. Biol. Chem. 272: 4637-44 (1997). Adaptor molecules may becritical in integrating multiple signaling cascades and in determiningthe cell type specific response to extracellular stimuli. These adaptorproteins have no apparent catalytic activity. Rather, they contain oneor more domains that mediate protein-protein or protein-lipidinteractions. The most common conserved interaction domains in theseadaptor molecules are Src homology (SH2), SH3, phosphotyrosine binding(PTB) and pleckstrin homology domains. [Reviewed in Pawson and Scott,Science 278: 2075-80 (1997)].

[0004] Signals generated by growth factors such as epidermal growthfactor (EGF) or insulin growth factor-1 (IGF-1) through receptortyrosine kinases (RTK) or by extracellular matrix components actingthrough the integrin receptors can induce cytoskeletal changes,Leventhal, et al., J. Biol. Chem. 272: 5214-18 (1997); Ojaniemi & Vuori,J. Biol. Chem. 272: 2443-47 (1996). There are also indications that RTKscan modulate integrin signals and vice versa, Doerr & Jones, J. Biol.Chem. 271: 2443-47 (1996); Jones et al., Proc. Natl. Acad. Sci USA 93:2482-87 (1996); Knight et al., J. Biol. Chem. 270: 10199-203 (1995);Matsumoto et al., Cancer Metas. Rev. 14: 205-17 (1995). However thedetails of how RTKs signal to the cytoskeletal components have not beenfully resolved. Further, while some adaptor proteins have a limitedpattern of expression [Liu & Roth, Proc. Natl. Acad. Sci. USA 92:10287-91 (1995); Nakamura et al., Oncogene 13: 1111-21 (1996)], many areubiquitously expressed [Araki et al., Diabetes 42: 1041-54 (1993);Frantz et al., J. Biol. Chem. 272: 2659-67 (1997)]. Thus, it is notclear how biologically relevant outputs are modulated as cellsdifferentiate.

[0005] Cancer is characterized by the increase in the number ofabnormal, or neoplastic, cells derived from a normal tissue whichproliferate to form a tumor mass, the invasion of adjacent tissues bythese neoplastic tumor cells, and the generation of malignant cellswhich eventually spread via the blood or lymphatic system to regionallymph nodes and to distant sites (metastasis). In a cancerous state acell proliferates under conditions in which normal cells would not grow.Cancer manifests itself in a wide variety of forms, characterized bydifferent degrees of invasiveness and aggressiveness.

[0006] Alteration of gene expression is intimately related to theuncontrolled cell growth and de-differentiation which are a commonfeature of all cancers. The genomes of certain well studied tumors havebeen found to show decreased expression of recessive genes, usuallyreferred to as tumor suppression genes, which would normally function toprevent malignant cell growth, and/or overexpression of certain dominantgenes, such as oncogenes, that act to promote malignant growth. Each ofthese genetic changes appears to be responsible for importing some ofthe traits that, in aggregate, represent the full neoplastic phenotype(Hunter, Cell 64: 1129 [1991]; Bishop, Cell 64: 235-248 [1991]).

[0007] A well known mechanism of gene (e.g. oncogene) overexpression incancer cells is gene amplification. This is a process where in thechromosome of the ancestral cell multiple copies of a particular geneare produced. The process involves unscheduled replication of the regionof chromosome comprising the gene, followed by recombination of thereplicated segments back into the chromosome (Alitalo et al., Adv.Cancer Res. 47: 235-281 [1986]). It is believed that the overexpressionof the gene parallels gene amplification, i.e. is proportionate to thenumber of copies made.

[0008] Proto-oncogenes that encode growth factors and growth factorreceptors have been identified to play important roles in thepathogenesis of various human malignancies, including breast cancer. Forexample, it has been found that the human ErbB2 gene (erbB2, also knownas her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoproteinreceptor (p185^(HER2); HER2) related to the epidermal growth factorreceptor EGFR), is overexpressed in about 25% to 30% of human breastcancer (Slamon et al., Science 235:177-182 [1987]; Slamon et al.,Science 244: 707-712 [1989]).

[0009] It has been reported that gene amplification of a proto-oncogeneis an event typically involved in the more malignant forms of cancer,and could act as a predictor of clinical outcome (Schwab et al., GenesChromosomes Cancer 1, 181-193 [1990]; Alitalo et al., supra). Thus,erbB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes (Slamon et al., [1987] and [1989], supra; Ravdinand Chamness, Gene 159: 19-27 [1995]; and Hynes and Stern, BiochemBiophys Acta 1198: 165-184 [1994]), and has been linked to sensitivityand/or resistance to hormone therapy and chemotherapeutic regimens,including CMF (cyclophosphamide, methotrexate, and fluoruracil) andanthracyclines (Baselga et al., Oncology 1 (3 Suppl 1): 43-48 [1997]).However, despite the association of erbB2 overexpression with poorprognosis, the odds of HER2-positive patients responding clinically totreatment with taxanes were greater than three times those ofHER2-negative patients. A recombinant humanized anti-ErbB2 (anti-HER2)monoclonal antibody (a humanized version of the murine anti-ErbB2antibody 4D5, referred to as rhuMAb HER2 or Herceptin®) has beenclinically active in patients with ErbB2-overexpressing metastaticbreast cancers that had received extensive prior anticancer therapy.(Baselga et al., J. Clin. Oncol. 14: 737-744 [1996]).

SUMMARY OF THE INVENTION

[0010] Applicants have identified cDNA clone (DNA30676, DNA40575,DNA61601)(SEQ ID NO:s 2, 4 & 6, respectively) that encodes a novelpolypeptide, designated in the present application as “Nsp1, Nsp2 &Nsp3” (SEQ ID NOS: 1, 3 and 6), respectively.

[0011] In one embodiment, the invention provides an isolated nucleicacid molecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a polypeptide comprising the sequence of amino acids 1to 576 of FIG. 1 (SEQ ID NO: 1), amino acids 1 to 501 of FIG. 2 (SEQ IDNO: 3) or amino acids 1 to 703 of FIG. 3 (SEQ ID NO: 5); or (b) thecomplement of the DNA molecule of (a). The sequence identity preferablyis about 85%, more preferably about 90%, most preferably about 95%. Inone aspect, the isolated nucleic acid has at least about 80%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% sequence identity with a polypeptidehaving amino acid residues 1 to 576 of FIG. 1 (SEQ ID NO: 1), 1 to 501of FIG. 2, (SEQ ID NO: 3), and 1 to 703 of FIG. 3 (SEQ ID NO: 5).Preferably, the greatest degree of identity occurs in the serine/prolinerich domain (i.e., amino acid residues 145-299 of SEQ ID NO: 1, aminoacid residues 28-210 of SEQ ID NO: 3 and amino acids 181-415 of SEQ IDNO: 5). Alternatively, the greatest degree of identity occurs in the SH2domain (i.e., amino acid residues 1-118 of SEQ ID NO:1 and amino acidresidues 50-166 of SEQ ID NO: 5).

[0012] In a further embodiment, the isolated nucleic acid moleculecomprises DNA encoding a PRO201, PRO308 or PRO309 polypeptide havingamino acid residues: (a) 1 to 576 of FIG. 1 (SEQ ID NO: 1), (b) 1 to 501of FIG. 2 (SEQ ID NO: 3), or (c) 1 to 703 of FIG. 3 (SEQ ID NO: 5); oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions.

[0013] Preferably, said nucleic acid molecule hybridizes to DNA encodinga fragment within the region defined by amino acid residues: (a) 32-576of FIG. 1 (SEQ ID NO:1) or (b) 1-424 or 506 to 703 of FIG. 3 (SEQ IDNO:5). In another aspect, the invention provides a nucleic acid of thefull length protein of clones DNA30676-1223 (SEQ ID NO:2), DNA40575-1223(SEQ ID NO:4) and DNA61601-1223 (SEQ ID NO:6), deposited with the ATCCunder accession number ATCC 209567, ATCC 209565 and ATCC 209713,respectively, alternatively the coding sequence of clones DNA30676-1223(SEQ ID NO:2), DNA40575-1223 (SEQ ID NO:4) and DNA61601-1223 (SEQ IDNO:6), deposited under accession number ATCC 209567, ATCC 209565 andATTC 209713, respectively.

[0014] In yet another embodiment, the invention provides a vectorcomprising DNA encoding a PRO201, PRO 308, or PRO309 polypeptide. A hostcell comprising such a vector is also provided. By way of example, thehost cells may be CHO cells, E. coli, or yeast. A process for producingPRO201, PRO308 or PRO309 polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of PRO201,PRO308 or PRO309 and recovering the same from the cell culture.

[0015] In yet another embodiment, the invention provides isolatedPRO201, PRO308 or PRO309 polypeptide. In particular, the inventionprovides isolated native sequence PRO201, PRO308 or PRO309 polypeptide,which in one embodiment, includes an amino acid sequence comprisingresidues 1 to 576 of FIG. 1 (SEQ ID NO: 1); 1 to 501 of FIG. 2 (SEQ IDNO: 3) or 1 to 703 of FIG. 3 (SEQ ID NO: 5). Native PRO201, PRO308 orPRO309 polypeptides with or without the initiating methionine arespecifically included. Alternatively, the invention provides a PRO201,PRO308 or PRO309 polypeptide encoded by the nucleic acid deposited underaccession number ATCC 209567, ATCC 209565 and ATTC209713, respectively.

[0016] In yet another embodiment, the invention provides chimericmolecules comprising a PRO201, PRO308 or PRO309 polypeptide fused to aheterologous polypeptide or amino acid sequence. An example of such achimeric molecule comprises a PRO201, PRO308 or PRO309 polypeptide fusedto an epitope tag sequence or an Fc region of an immunoglobulin.

[0017] In yet another embodiment, the invention provides for compoundsand methods for developing antagonists against and agonists promotingthe PRO201, PRO308 and/or PRO309 modulated cellular signaling. Inparticular, an antagonist of PRO201 (e.g., Nsp1, SEQ ID NO:1), PRO308(e.g., Nsp2, SEQ ID NO:3) and/or PRO309 (e.g., Nsp3, SEQ ID NO:5) whichblocks, inhibits and/or neutralizes the normal functioning of the lattercompounds in cellular signaling, including both small bioorganicmolecules and antisense nucleotides.

[0018] In yet another embodiment, the invention provides foralternatively spliced variants of PRO201, PRO308 or PRO309 (e.g.,DNA40556).

[0019] The present invention further concerns compositions and methodsfor the diagnosis and treatment of neoplastic cell growth andproliferation in mammals, including humans. The present invention isbased on the identification of genes that are amplified in the genome oftumor cells. Such gene amplification is expected to be associated withthe overexpression of the gene product and contribute to tumorigenesis.Accordingly, the proteins encoded by the amplified genes are believed tobe useful targets for the diagnosis and/or treatment (includingprevention) of certain cancers, and may act of predictors of theprognosis of tumor treatment.

[0020] In one embodiment, the present invention concerns an isolatedantibody which binds a polypeptide which is designated PRO201 (e.g.,Nsp1, SEQ ID NO:1), PRO308 (e.g., Nsp2, SEQ ID NO:3) or PRO309 (e.g.,Nsp3, SEQ ID NO:5). In one aspect, the antibody induces death of a celloverexpressing a PRO201, PRO308 or PRO309 polypeptide. In anotheraspect, the antibody is a monoclonal antibody, which preferably hasnonhuman complementarity determining region (CDR) residues and humanframework region (FR) residues. The antibody may be labeled and may beimmobilized on a solid support. In a further aspect, the antibody is anantibody fragment, a single-chain antibody, or an anti-idiotypicantibody.

[0021] In another embodiment, the invention concerns a compositioncomprising an antibody which binds a PRO201, PRO308 or PRO309polypeptide in admixture with a pharmaceutically acceptable carrier. Inone aspect, the composition comprises a therapeutically effective amountof the antibody. In another aspect, the composition comprises a furtheractive ingredient, which may, for example, be a further antibody or acytotoxic or chemotherapeutic agent. Preferably, the composition issterile.

[0022] In a further embodiment, the invention concerns nucleic acidencoding an anti-PRO201, anti-PRO308 or anti-PRO309 antibody, andvectors and recombinant host cells comprising such nucleic acid.

[0023] In a still further embodiment, the invention concerns a methodfor producing an anti-PRO201, anti-PRO308 or anti-PRO309 antibody byculturing a host cell transformed with nucleic acid encoding theantibody under conditions such that the antibody is expressed, andrecovering the antibody from the cell culture.

[0024] The invention further concerns antagonists and agonists of aPRO201, PRO308 or PRO309 polypeptide that inhibit one or more of thefunctions or activities of the PRO201, PRO308 or PRO309 polypeptide.

[0025] In a further embodiment, the invention concerns isolated nucleicacid molecules that hybridize to the complement of the nucleic acidmolecules encoding the PRO201, PRO308 or PRO309 polypeptides. Thenucleic acid preferably is DNA, and hybridization preferably occursunder stringent conditions. Preferably, said nucleic acid moleculehybridizes to the region from nucleotide residues (a) about 245 to 2413of FIG. 1 (SEQ ID NO:2) or (b) 1 to about 1312 or about 1555 to about2150 of FIG. 3 (SEQ ID NO:6). Such nucleic acid molecules can act asantisense molecules of the amplified genes identified herein, which, inturn, can find use in the modulation of the respective amplified genes,or as antisense primers in amplification reactions. Furthermore, suchsequences can be used as part of ribozyme and/or triple helix sequencewhich, in turn, may be used in regulation of the amplified genes.

[0026] In another embodiment, the invention concerns a method fordetermining the presence of a PRO201, PRO308 or PRO309 polypeptidecomprising exposing a cell suspected of containing the PRO201, PRO308 orPRO309 polypeptide to an anti-PRO201, anti-PRO308 or anti-PRO309antibody and determining binding of the antibody to the cell.

[0027] In yet another embodiment, the present invention concerns amethod of diagnosing tumor in a mammal, comprising detecting the levelof expression of a gene encoding a PRO201, PRO308 or PRO309 polypeptide(a) in a test sample of tissue cells obtained from the mammal, and (b)in a control sample of known normal tissue cells of the same cell type,wherein a higher expression level in the test sample indicates thepresence of tumor in the mammal from which the test tissue cells wereobtained.

[0028] In another embodiment, the present invention concerns a method ofdiagnosing tumor in a mammal, comprising (a) contacting an anti-PRO201,anti-PRO308 or anti-PRO309 antibody with a test sample of tissue cellsobtained from the mammal, and (b) detecting the formation of a complexbetween the anti-PRO201, anti-PRO308 or anti-PRO309 antibody and thePRO201, PRO308 or PRO309 polypeptide in the test sample. The detectionmay be qualitative or quantitative, and may be performed in comparisonwith monitoring the complex formation in a control sample of knownnormal tissue cells of the same cell type. A larger quantity ofcomplexes formed in the test sample indicates the presence of tumor inthe mammal from which the test tissue cells were obtained. The antibodypreferably carries a detectable label. Complex formation can bemonitored, for example, by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art.

[0029] The test sample is usually obtained from an individual suspectedto have neoplastic cell growth or proliferation (e.g. cancerous cells).

[0030] In another embodiment, the present invention concerns a cancerdiagnostic kit, comprising an anti-PRO201, anti-PRO308 or anti-PRO309antibody and a carrier (e.g. a buffer) in suitable packaging. The kitpreferably contains instructions for using the antibody to detect thePRO201, PRO308 or PRO309 polypeptide.

[0031] In yet another embodiment, the invention concerns a method forinhibiting the growth of tumor cells comprising exposing a cell whichoverexpresses a PRO201, PRO308 or PRO309 polypeptide to an effectiveamount of an agent inhibiting the expression and/or activity of thePRO201, PRO308 or PRO309 polypeptide. The agent preferably is ananti-PRO201, anti-PRO308 or anti-PRO309 antibody, a small organic andinorganic molecule, peptide, phosphopeptide, antisense or ribozymemolecule, or a triple helix molecule. In a specific aspect, the agent,e.g. anti-PRO201, anti-PRO308 or anti-PRO309 antibody induces celldeath. In a further aspect, the tumor cells are further exposed toradiation treatment and/or a cytotoxic or chemotherapeutic agent.

[0032] In a further embodiment, the invention concerns an article ofmanufacture, comprising:

[0033] a container;

[0034] a label on the container; and

[0035] a composition comprising an active agent contained within thecontainer; wherein the composition is effective for inhibiting thegrowth of tumor cells, the label on the container indicates that thecomposition can be used for treating conditions characterized byoverexpression of a PRO201, PRO308 or PRO309 polypeptide, and the activeagent in the composition is an agent inhibiting the expression and/oractivity of PRO201, PRO308 or PRO309 polypeptide. In a preferred aspect,the active agent is an anti-PRO201, anti-PRO308 or anti-PRO309 antibody.

[0036] A method for identifying a compound capable of inhibiting theexpression and/or activity of a PRO201, PRO308 or PRO309 polypeptide,comprising contacting a candidate compound with a PRO201, PRO308 orPRO309 polypeptide under conditions and for a time sufficient to allowthese two components to interact. In a specific aspect, either thecandidate compound or the PRO201, PRO308 or PRO309 polypeptide isimmobilized on a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1 shows both the nucleic acid sequence of DNA30676 (SEQ IDNO:2) as well as the encoded amino acid sequence of a native sequencePRO201 (Nsp1) polypeptide (SEQ ID NO:1).

[0038]FIG. 2 shows both the nucleic acid sequence of DNA40575 (SEQ IDNO:4) as well as the encoded amino acid sequence of a native sequencePRO308 (Nsp2) polypeptide (SEQ ID NO:3).

[0039]FIG. 3 show both the nucleic acid sequence of DNA61601 (SEQ IDNO:6) as well as the encoded amino acid sequence of a native sequencePRO309 (Nsp3) polypeptide (SEQ ID NO:5).

[0040] FIGS. 4A-C shows the sequences of 1328938 (SEQ ID NO:13), 104191(SEQ ID NO: 14) and 1651811 (SEQ ID NO: 15), respectively, of the(LIFESEQ® database, Incyte Pharmaceuticals, Palo Alto, Calif.), whichwere used to isolate the full length DNA30676 (SEQ ID NO:2), DNA40575(SEQ ID NO:4) and DNA61601 (SEQ ID NO:6) nucleic acid sequences of theinvention.

[0041]FIG. 5A shows the oligonucleotide sequences (SEQ ID NO: 7, SEQ IDNO: 8) which were used in the isolation of DNA30676 (SEQ ID NO:2). FIG.5B shows the oligonucleotide sequences (SEQ ID NO: 9, SEQ ID NO: 10)which were used in the isolation of DNA40575 (SEQ ID NO:4). FIG. 5Cshows the oligonucleotide sequences (SEQ ID NO: 11, SEQ ID NO: 12) whichwere used in the isolation of DNA61601 (SEQ ID NO:6).

[0042]FIG. 6A shows a figurative illustrative comparison of the variousdomains between Nsp1 (SEQ ID NO:1), Nsp2 (SEQ ID NO:3) and Nsp3 (SEQ IDNO:5). FIG. 6B show an actual comparison between the 3 sequencesthemselves introducing gaps, as necessary in order to maximize theoverall degree of identity between the three sequences.

[0043]FIG. 7 shows the sequence of Nsp1 (SEQ ID NO:1) wherein the SH2region is identified in bold and the prolines and serines of the P/Sregion are indicated in single and double underline, respectively.

[0044]FIG. 8 shows a comparison of Nsp1 (SEQ ID NO:1) with human Shc(SEQ ID NO:16), Sck (SEQ ID NO: 17) and Fes (SEQ ID NO: 18) proteins.

[0045]FIG. 9A is a northern blot showing significant expression of Nsp1(SEQ ID NO:2) in human fetal liver, while Nsp2 (SEQ ID NO:4) and Nsp3(SEQ ID NO:6) were more widely expressed. FIG. 9B shows the expressionof two Nsp3 transcripts in hematopoietic tissues.

[0046]FIGS. 10A and 10C are western blots wherein anti-flagimmunoprecipitates were blotted with anti-flag, anti-(P)Tyr or anti-Casantibodies as indicated. In FIG. 10B, anti-EGF receptor (CalBiochem)immunoprecipitates were blotted with anti-flag or anti-(P)Tyr antibodiesas indicated.

[0047]FIG. 11 is a western blot showing immunoprecipitates withanti-Flag or anti-p130^(Cas) and blotting with anti-(P)Tyr Ab PY-20 oranti-p 130^(Cas).

[0048]FIG. 12A is a western blot showing reduced phosphorylation of Nsp1(SEQ ID NO:1) upon treatment with insulin. FIG. 12B is western blotcreated by stripping the blot in FIG. 12A which was reprobed withanti-p130^(Cas) to confirm that the 130 kD protein was in factp130^(Cas).

[0049]FIG. 13 is a western blot of various Nsp1 mutants which have beentransfected into COS cells and treated with EGF, and the cell lysatesimmunoprecipitated with anti-flag Ab and Western blotted with eitheranti-(P)Tyr, anti-p130^(Cas) or the anti-flag Ab.

[0050]FIG. 14A is a micrograph of an ultrathin section ofretroviral-infected vector MSCV NIH3T3 cells, while FIG. 14B show Nsp1(SEQ ID NO:2) transfected NIH3T3 cells. FIG. 14C is a micrograph of anultrathin section of four week tumors which were fixed, blocked andsections stained with hematoxylin and eosin.

[0051]FIG. 15 is a bar graph of tumor size comparing vector controltransfection and Nsp1.sc1, .sc2 and .sc3 cells.

[0052]FIG. 16 is a bar graph of showing resistance to apoptosis ofgrowth factor deprived NIH3T3 of transformed subclones Nsp.sub1.1,Nsp.sub.2.1, nontransformed cell culture (NIH3T3-Nsp1.non-trans) and thecontrol cells NIH3T3-neo.

[0053]FIG. 17 is a western blot of COS cells transfected with pRK orNsp1, treated with EGF or no treatment, which were lysed in CoIP bufferand incubated with PI3-kinase N-terminal or C-terminal SH2 domain-GSTbeads (UBI). The precipitated Nsp1 was detected with anti-flag antibody.

[0054]FIG. 18 is a pictoral representation of chromosome 19 depictingthe rough approximation of the mapping of DNA30676 (SEQ ID NO:2) in thehuman genome.

[0055]FIG. 19 shows DNA40556 (SEQ ID NO:20) a splice variant of DNA30676(SEQ ID NO:2) which was also found to be amplified in various lung andcolon tumors.

[0056]FIG. 20 is a comparison of Nsp1 (SEQ ID NO:2), Nsp2 (SEQ ID NO:4)and the protein sequence encoded by DNA40556 (SEQ ID NO:20).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] I. Definitions

[0058] The terms “PRO201, PRO308 or PRO309,” “PRO201, PRO308 or PRO309polypeptide”, when used herein encompass both native sequence PRO201,PRO308 or PRO309, respectively, and variants thereof (which are furtherdefined herein). They may be isolated from a variety of sources, such asfrom human tissue types or from another source, or prepared byrecombinant or synthetic methods.

[0059] A “native sequence PRO201, PRO308 or PRO309” comprises apolypeptide having the same amino acid sequence as a PRO201, PRO308 orPRO309 derived from nature. Such native sequence PRO201, PRO308 orPRO309 can be isolated from nature or can be produced by recombinant orsynthetic means. The term “native sequence PRO201, PRO308 or PRO309”specifically encompasses naturally-occurring truncated or secreted formsof PRO201, PRO308 or PRO309 (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of PRO201, PRO308 or PRO309.Throughout this specification, the “PRO” and “Nsp” designations are bothused to designate the respective proteins, wherein “Nsp” designates onlythe particular native human sequence of the sequence of FIGS. 1, 2, 3and/or the sequence of the cDNA insert deposited under ATCC 209567,209565 and 209713, respectively. Whereas “Nsp” designates theparticularly enumerated species, “PRO” designates the native sequence(including the Nsps) and active variants thereof. In one embodiment ofthe invention, the native sequence PRO201, PRO308 or PRO309 is a matureor full-length native sequence PRO201, PRO308 or PRO309 comprising: (a)amino acids 1 to 576 of FIG. 1 (SEQ ID NO: 1); (b) amino acids 1 to 501of FIG. 2 (SEQ ID NO: 3) and (c) amino acids 1 to 703 of FIG. 3 (SEQ IDNO: 5), respectively, with or without the N-terminal signal sequence,and with or without the initiating methionine at position 1.

[0060] “PRO201, PRO308 or PRO309 variant” means a biologically activePRO201, PRO308 or PRO309, respectively, as defined below having at leastabout 80% amino acid sequence identity to: (a) a DNA molecule encoding aPRO201, PRO308 or PRO309 polypeptide, with or without its native signalsequence, or (b) the complement of the DNA molecule of (a). In aparticular embodiment, the PRO201, PRO308 or PRO309 variant has at leastabout 80% amino acid sequence homology with the PRO201, PRO308 or PRO309having the deduced amino acid sequence shown in FIG. 1 (SEQ ID NO: 1,FIG. 2 (SEQ ID NO: 3) or FIG. 3 (SEQ ID NO: 5) for a full-length nativesequence PRO201, PRO308 or PRO309. Such PRO201, PRO308 or PRO309variants include, for instance, PRO201, PRO308 or PRO309 polypeptideswherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the sequence of FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ IDNO: 3) or FIG. 3 (SEQ ID NO: 5). Preferably, the nucleic acid or aminoacid sequence identity is at least about 85%, more preferably at leastabout 90%, and even more preferably at least about 95%.

[0061] “Percent (%) amino acid sequence identity” with respect to thePRO201, PRO308 or PRO309 sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the PRO201, PRO308 or PRO309sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST-2 software set to the default parameters. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. Alternatively, thepercentage identity values used herein may be generated by the program“Align-2”, which has no parameter settings, was authored by Genentech,Inc. and which was filed on Dec. 10, 1991 with user documentation in theUnited States Copyright Office, Washington, D.C. 20559. Align-2 isregistered under U.S. copyright registration number TXU-510087.

[0062] “Percent (%) nucleic acid sequence identity” with respect to thePRO201, PRO308 or PRO309 sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO201, PRO308 or PRO309 sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST-2 software set thedefault parameters. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. Alternatively, the percentage identity values used herein maybe generated by the program “Align-2”, which has no parameter settings,was authored by Genentech, Inc. and which was filed on Dec. 10, 1991with user documentation in the United States Copyright Office,Washington, D.C. 20559. Align-2 is registered under U.S. copyrightregistration number TXU-510087.

[0063] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO201, PRO308 orPRO309 natural environment will not be present. Ordinarily, however,isolated polypeptide will be prepared by at least one purification step.

[0064] An “isolated” DNA30676 (SEQ ID NO:2), DNA40575 (SEQ ID NO:4) orDNA61601 (SEQ ID NO:6) nucleic acid molecule is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the DNA30676, DNA40575 or DNA61601 nucleic acid (SEQ ID NO:s2, 4 & 6, respectively). An isolated DNA30676, DNA40575 or DNA61601nucleic acid molecule (SEQ ID NO:s 2, 4 & 6, respectively) is other thanin the form or setting in which it is found in nature. IsolatedDNA30676, DNA40575 or DNA61601 nucleic acid molecules (SEQ ID NO:s 2, 4& 6, respectively) therefore are distinguished from the DNA30676,DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO:s 2, 4 & 6,respectively) as it exists in natural cells. However, an isolatedDNA30676, DNA40575 or DNA61601 nucleic acid molecule (SEQ ID NO:s 2, 4 &6, respectively) includes DNA30676, DNA40575 or DNA61601 nucleic acidmolecules (SEQ ID NO:s 2, 4 & 6, respectively) contained in cells thatordinarily express DNA30676, DNA40575 or DNA61601 (SEQ ID NO:s 2, 4 & 6,respectively) where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

[0065] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0066] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0067] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like. Theterm “epitope tagged” when used herein refers to a chimeric polypeptidecomprising a DNA30676, DNA40575 or DNA61601 polypeptide (SEQ ID NO:1, 3& 5, respectively) fused to a “tag polypeptide”. The tag polypeptide hasenough residues to provide an epitope against which an antibody can bemade, yet is short enough such that it does not interfere with activityof the polypeptide to which it is fused. The tag polypeptide preferablyalso is fairly unique so that the antibody does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least six amino acid residues and usually between about 8 and 50amino acid residues (preferably, between about 10 and 20 amino acidresidues).

[0068] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0069] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0070] The term “antibody” is used in the broadest sense andspecifically covers single anti-PRO201, anti-PRO308 or anti-PRO309monoclonal antibodies (including agonist, antagonist, and neutralizingantibodies) and anti-PRO201, anti-PRO308 or anti-PRO309 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

[0071] “Active” or “activity” in the context of molecules identifiedbased upon the PRO201, PRO308 or PRO309 polypeptides (or their codingsequences) refers to polypeptides (e.g., antibodies) or organic orinorganic small molecules, peptides, etc. which retain a biologicaland/or immunological activity of native or naturally-occurring PRO201,PRO308 or PRO309. A “biological” activity refers to a biologicalfunction (either inhibitory or stimulatory), caused by a native ornaturally-occurring PRO201, PRO308 or PRO309, other than the ability toinduce the production of an antibody against an antigenic epitopepossessed by a native or naturally-occurring PRO201, PRO308 or PRO309.An “immunological” activity refers to the ability to induce theproduction of an antibody against an antigenic epitope possessed by anative or naturally occurring PRO201, PRO308 or PRO309.

[0072] A preferred biological activity includes, for example, abiological activity identified by the screening assays identifiedherein, for example, growth inhibition of a target tumor cell. Anotherpreferred biological activity is cytotoxic activity resulting in thedeath of the target tumor cell. Additional preferred activities involvethe regulation or tumorigenesis and response to stimulation by, forexample, integrin receptors ligands and by epidermal growth factor(EGF), insulin growth factor (IGF) and through other receptor tyrosine(RTK) ligands. More preferably, biological activity can be determined bymeasurement of guanylate exchange, in particular, the activation ofc-jun kinase (JNK).

[0073] The term “modulate” means to affect (e.g., either upregulate,downregulate or otherwise control) the level of a signaling pathway.Cellular processes under the control of signal transduction include, butare not limited to, transcription of specific genes, normal cellularfunctions, such as metabolism, proliferation, differentiation, adhesion,apoptosis and survival, as well as abnormal processes, such astransformation, blocking of differentiation and metastasis.

[0074] The term “antagonist” is used herein in the broadest sense toinclude any molecule which blocks, prevents, inhibits, or neutralizesthe process by which the PRO201, PRO308 or PRO309 molecules of theinvention that interferes with the interaction of any of the proteindomains of PRO201, PRO308 or PRO309 with various target proteins. Suchinteractions can generally occur with the C-terminal end of PRO201,PRO308 or PRO309, or specifically the SH2 and/or proline/serine (P/S)rich regions with phosphotyrosyl residues and polyproline motifs with atarget binding site. In a similar manner, the term “agonist” is usedherein to include any molecule which promotes, enhances or stimulatesthe interaction of the protein domains of PRO201, PRO308 or PRO309(e.g., Nsp1, Nsp2 and Nsp3, respectively) including the SH2 and/orproline/serine (P/S) rich regions with phosphotyrosyl residues andpolyproline motifs, respectively on various target proteins. Suitablemolecules that affect the interaction of the SH2 and/or P/S regions ofPRO201, PRO308 or PRO309 and the phosphotyrosyl residues and polyprolinemotifs, respectively or target proteins include fragments of the latteror small bioorganic molecules, e.g., peptidomimetics, which will preventor enhance, as the case may be, the interaction. Non-limiting examplesinclude proteins, peptides, glycoproteins, glycopeptides, glycolipids,polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules,peptidomimetics, pharmacological agents and their metabolites,transcriptional and translation control sequences, and the like. Anotherpreferred form of antagonist includes antisense nucleotides that inhibitthe PRO201, PRO308 or PRO309 modulated signaling. Preferred forms bindto specific regions on either PRO201, PRO308 or PRO309 or the targetswith which PRO201, PRO308 or PRO309 interact.

[0075] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons. “Antibodies” (Abs) and “immunoglobulins” (Igs)are glycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity. Polypeptides of the latterkind are, for example, produced at low levels by the lymph system and atincreased levels by myelomas. The term “antibody” is used in thebroadest sense and specifically covers, without limitation, intactmonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g. bispecific antibodies) formed from at least two intact antibodies,and antibody fragments so long as they exhibit the desired biologicalactivity.

[0076] The phrases “gene amplification” and “gene duplication” are usedinterchangeably and refer to a process by which multiple copies of agene or gene fragment are formed in a particular cell or cell line. Theduplicated region (a stretch of amplified DNA) is often referred to as“amplicon.” Usually, the amount of the messenger RNA (mRNA) produced,i.e. the level of gene expression, also increases in the proportion ofthe number of copies made of the particular gene expressed.

[0077] “Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

[0078] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

[0079] “Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g. cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g radiation and/or chemotherapy.

[0080] The “pathology” of cancer includes all phenomena that compromisethe well-being of the patient. This includes, without limitation,abnormal or uncontrollable cell growth, metastasis, interference withthe normal functioning of neighboring cells, release of cytokines orother secretory products at abnormal levels, suppression or aggravationof inflammatory or immunological response, etc.

[0081] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, hamsters, rats,mice, cattle, pigs, goats, sheep, etc. Preferably, the mammal is human.

[0082] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0083] Administration “in combination with” or “admixture with” one ormore further therapeutic agents includes simultaneous (concurrent) andconsecutive administration in any order.

[0084] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g. I¹³¹, I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fungal, plantor animal origin, or fragments thereof.

[0085] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g. paclitaxel (Taxol™, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (see U.S.Pat. No. 4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycinD, VP-16, chlorambucil, melphalan, and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

[0086] A “growth inhibitory agent” when used herein refers to a compoundor composition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (W BSaunders: Philadelphia, 1995), especially p. 13.

[0087] “Doxorubicin” is an athracycline antibiotic. The full chemicalname of doxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

[0088] The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon -α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TGF-αand TGF-β; and other polypeptide factors including LIF and kit ligand(KL). As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

[0089] The term “prodrug” as used in this application refers to aprecursor or derivative form of a pharmaceutically active substance thatis less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive parent form. See, e.g. Wilman, “Prodrugs in Cancer Chemotherapy”,Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting,Belfast (1986), and Stella et al., “Prodrugs: A Chemical Approach toTargeted Drug Delivery”, Directed Drug delivery, Borchardt et al.,(ed.), pp. 147-267, Humana Press (1985). The prodrugs of this inventioninclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glysocylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrugs form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

[0090] “Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 Dalton, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

[0091] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three or four segments called“complementarity-determining regions” (CDRs) or “hypervariable regions”in both in the light-chain and the heavy-chain variable domains. Themore highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four or five FR regions, largely adopting a β-sheetconfiguration, connected by the CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., NIH PubI.No.91-3242, Vol. 1, pages 647-669 (1991)). The constant domains are notnecessarily involved directly in binding an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

[0092] The term “hypervariable region” or “complementarity-determiningregions” (CDRs) as used herein define a subregion within the variableregion of extreme sequence variability of the antibody, which form theantigen-binding site and are the main determinants of antigenspecificity. According to one definition, they can be residues (Kabatnomenclature) 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable region and residues (Kabat nomenclature 31-35 (H1), 50-65 (H2),95-102 (H3) in the heavy chain variable region. Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institute of Health, Bethesda, Md. [19911). Alternatively, orin combination with the region defined by Kabat, the hypervariableregion can be the “hypervariable loop”, comprising residues (Chothianomenclature) 26-32 (L1), 50-53 (L2), 91-96 (L3) in the light chainvariable region and residue (Chothia nomenclature) 26-32 (H1), 53-55(L2) and 96-101 (L3); Chothia and Lesk, J. Mol. Biol. 196: 901-917[1987]). “Framework” or “FR” residues are those variable domain residuesof relatively low sequence variability which lie in between the CDRregions.

[0093] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8 (10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0094] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen.

[0095] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0096] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fabfragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

[0097] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains.

[0098] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

[0099] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al., Nature, 256:495 [1975], or maybe made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352: 624-628 [1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

[0100] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

[0101] “Humanized” forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv FR residuesof the human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andmaximize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332: 323-329 [1988]; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a PRIMATIZED™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

[0102] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0103] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0104] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0105] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.Radionuclides that can serve as detectable labels include, for example,I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, andPd-109.

[0106] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

[0107] II. Compositions and Methods of the Invention

[0108] A. Full-length PRO201, PRO308 or PRO309

[0109] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO201 (e.g., Nsp1, SEQ ID NO:1), PRO308 (e.g., Nsp2, SEQID NO:3) or PRO309 (e.g., Nsp3, SEQ ID NO:5). In particular, Applicantshave identified and isolated cDNA encoding a PRO201, PRO308 and PRO309polypeptide, as disclosed in further detail in the Examples below. Usingthe BLAST-2 sequence alignment computer program set to the defaultparameters, Applicants found that a full-length native sequence Nsp1 andNsp3 (shown in FIGS. 6A & 6B and SEQ ID NO:1 & 3, respectively) haveregions of SH2 homology, while Nsp1, Nsp2 and Nsp3 (SEQ ID NO: 1, 3 & 5)have a proline/serine rich (P/S) region homology. SH2 domains are knownto bind specific phosphotyrosyl residues, while the P/S region could bea potential SH3 interaction domain. Accordingly, it is presentlybelieved that PRO201, PRO308 and PRO309 disclosed in the presentapplication are newly identified family of adaptor proteins and maypossess properties which modulate intracellular signaling pathways.

[0110] B. PRO201, PRO308 or PRO309 Variants

[0111] In addition to the full-length native sequence PRO201, PRO308 andPRO309 described herein, it is contemplated that PRO201, PRO308 andPRO309 variants can be prepared. PRO201, PRO308 and PRO309 variants canbe prepared by introducing appropriate nucleotide changes into thePRO201, PRO308 or PRO309 DNA, or by synthesis of the desired PRO201,PRO308 or PRO309 polypeptides. Those skilled in the art will appreciatethat amino acid changes may alter post-translational processes of thePRO201, PRO308 or PRO309, such as changing the number or position ofglycosylation sites or altering the membrane anchoring characteristics.

[0112] Variations in the native full-length sequence PRO201, PRO308 orPRO309 or in various domains of the PRO201, PRO308 or PRO309 describedherein, can be made, for example, using any of the techniques andguidelines for conservative and non-conservative mutations set forth,for instance, in U.S. Pat. No. 5,364,934. Variations may be asubstitution, deletion or insertion of one or more codons encoding thePRO201, PRO308 or PRO309 that results in a change in the amino acidsequence of the PRO201, PRO308 or PRO309 as compared with the nativesequence PRO201, PRO308 or PRO309. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the PRO201, PRO308 or PRO309. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO201, PRO308 or PRO309 with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to 5 amino acids. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activity inthe in vitro assay described in the Examples below.

[0113] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO201, PRO308 or PRO309 variant DNA.

[0114] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

[0115] C. Modifications of PRO201, PRO308 or PRO309

[0116] Covalent modifications of PRO201, PRO308 or PRO309 are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of the PRO201, PRO308 orPRO309 with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues of thePRO201, PRO308 or PRO309. Derivatization with bifunctional agents isuseful, for instance, for crosslinking PRO201, PRO308 or PRO309 to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO201, PRO308 or PRO309 antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

[0117] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0118] Another type of covalent modification of the PRO201, PRO308 orPRO309 polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencePRO201, PRO308 or PRO309, and/or adding one or more glycosylation sitesthat are not present in the native sequence PRO201, PRO308 or PRO309,and/or alteration of the ratio and/or composition of the sugar residuesattached to the glycosylation site(s).

[0119] Addition of glycosylation sites to the PRO201, PRO308 or PRO309polypeptide may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequencePRO201, PRO308 or PRO309 (for O-linked glycosylation sites). The PRO201,PRO308 or PRO309 amino acid sequence may optionally be altered throughchanges at the DNA level, particularly by mutating the DNA encoding thePRO201, PRO308 or PRO309 polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

[0120] Another means of increasing the number of carbohydrate moietieson the PRO201, PRO308 or PRO309 polypeptide is by chemical or enzymaticcoupling of glycosides to the polypeptide. Such methods are described inthe art, e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0121] Removal of carbohydrate moieties present on the PRO201, PRO308 orPRO309 polypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0122] Another type of covalent modification of PRO201, PRO308 or PRO309comprises linking the PRO201, PRO308 or PRO309 polypeptide to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

[0123] The PRO201, PRO308 or PRO309 of the present invention may also bemodified in a way to form a chimeric molecule comprising PRO201, PRO308or PRO309 fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof the PRO201, PRO308 or PRO309 with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino- or carboxyl-terminus of thePRO201, PRO308 or PRO309. The presence of such epitope-tagged forms ofthe PRO201, PRO308 or PRO309 can be detected using an antibody againstthe tag polypeptide. Also, provision of the epitope tag enables thePRO201, PRO308 or PRO309 to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. In an alternative embodiment, the chimeric moleculemay comprise a fusion of the PRO201, PRO308 or PRO309 with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule, such a fusion could be to the Fcregion of an IgG molecule.

[0124] Various tag polypeptides and their respective antibodies are wellknown in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0125] D. Preparation of PRO201, PRO308 or PRO309

[0126] The description below relates primarily to production of PRO201,PRO308 OR PRO309 by culturing cells transformed or transfected with avector containing PRO201, PRO308 OR PRO309 nucleic acid. It is, ofcourse, contemplated that alternative methods, which are well known inthe art, may be employed to prepare PRO201, PRO308 or PRO309. Forinstance, the PRO201, PRO308 or PRO309 sequence, or portions thereof,may be produced by direct peptide synthesis using solid-phase techniques[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W. H. FreemanCo., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the PRO201, PRO308 or PRO309 may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length PRO201, PRO308 or PRO309.

[0127] 1. Isolation of DNA Encoding PRO201, PRO308 or PRO309

[0128] DNA encoding PRO201, PRO308 or PRO309 may be obtained from a cDNAlibrary prepared from tissue believed to possess the PRO201, PRO308 orPRO309 mRNA and to express it at a detectable level. Accordingly, humanPRO201, PRO308 or PRO309 DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue, such as described in the Examples.The PRO201, PRO308 or PRO309-encoding gene may also be obtained from agenomic library or by oligonucleotide synthesis.

[0129] Libraries can be screened with probes (such as antibodies to thePRO201, PRO308 or PRO309 or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding PRO201, PRO308 or PRO309 is to use PCR methodology [Sambrook etal., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1995)].

[0130] Exemplary techniques for screening a cDNA library are describedbelow in the Examples. The oligonucleotide sequences selected as probesshould be of sufficient length and sufficiently unambiguous that falsepositives are minimized. The oligonucleotide is preferably labeled suchthat it can be detected upon hybridization to DNA in the library beingscreened. Methods of labeling are well known in the art, and include theuse of radiolabels like ³²P-labeled ATP, biotinylation or enzymelabeling. Hybridization conditions, including moderate stringency andhigh stringency, are provided in Sambrook et al., supra.

[0131] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined through sequence alignment using computer softwareprograms such as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT whichemploy various algorithms to measure homology.

[0132] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0133] 2. Selection and Transformation of Host Cells

[0134] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO201, PRO308 or PRO309 productionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0135] Methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Depending on the hostcell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0136] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635).

[0137] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forPRO201, PRO308 OR PRO309-encoding vectors. Saccharomyces cerevisiae is acommonly used lower eukaryotic host microorganism.

[0138] Suitable host cells for the expression of glycosylated PRO201,PRO308 or PRO309 are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor (MMT 060562, ATCC CCL51). The selection of the appropriate hostcell is deemed to be within the skill in the art.

[0139] 3. Selection and Use of a Replicable Vector

[0140] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO201,PRO308 or PRO309 may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

[0141] The PRO201, PRO308 or PRO309 may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO201, PRO308 or PRO309 DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published Apr. 4, 1990), or the signal described inWO 90/13646 published Nov. 15, 1990. In mammalian cell expression,mammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders.

[0142] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0143] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0144] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePRO201, PRO308 or PRO309 nucleic acid, such as DHFR or thymidine kinase.An appropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

[0145] Expression and cloning vectors usually contain a promoteroperably linked to the PRO201, PRO308 or PRO309 nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgamo (S.D.) sequence operably linked to the DNA encodingPRO201, PRO308 or PRO309.

[0146] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem, 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0147] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0148] PRO201, PRO308 or PRO309 transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtained from thegenomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, and from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

[0149] Transcription of a DNA encoding the PRO201, PRO308 or PRO309 byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO201, PRO308 or PRO309 coding sequence, but is preferably located at asite 5′ from the promoter.

[0150] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding PRO201, PRO308 or PRO309.

[0151] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of PRO201, PRO308 or PRO309 in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

[0152] 4. Detecting Gene Amplification/Expression

[0153] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0154] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO201, PRO308 or PRO309 polypeptide or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to PRO201, PRO308 or PRO309 DNA and encoding a specific antibodyepitope.

[0155] 5. Purification of Polypeptide

[0156] Forms of PRO201, PRO308 or PRO309 may be recovered from culturemedium or from host cell lysates. If membrane-bound, it can be releasedfrom the membrane using a suitable detergent solution (e.g. Triton-X100) or by enzymatic cleavage. Cells employed in expression of PRO201,PRO308 or PRO309 can be disrupted by various physical or chemical means,such as freeze-thaw cycling, sonication, mechanical disruption, or celllysing agents.

[0157] It may be desired to purify PRO201, PRO308 or PRO309 fromrecombinant cell proteins or polypeptides. The following procedures areexemplary of suitable purification procedures: by fractionation on anion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of the PRO201, PRO308 or PRO309. Variousmethods of protein purification may be employed and such methods areknown in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO201, PRO308 or PRO309 produced.

[0158] E. Uses for PRO201, PRO308 OR PRO309

[0159] Nucleotide sequences (or their complement) encoding PRO201,PRO308 or PRO309 have various applications in the art of molecularbiology, including uses as hybridization probes, in chromosome and genemapping and in the generation of anti-sense RNA and DNA. PRO201, PRO308or PRO309 nucleic acid will also be useful for the preparation ofPRO201, PRO308 or PRO309 polypeptides by the recombinant techniquesdescribed herein.

[0160] The full-length native sequence PRO201, PRO308 or PRO309 (SEQ IDNO:1, 3 & 5) gene, or portions thereof, may be used as hybridizationprobes for a cDNA library to isolate the full-length gene or to isolatestill other genes (for instance, those encoding naturally-occurringvariants of PRO201, PRO308 or PRO309 or PRO201, PRO308 or PRO309 fromother species) which have a desired sequence identity to the PRO201,PRO308 or PRO309 sequence disclosed in FIGS. 1-3 (SEQ ID NO: 1, 3 & 5).Optionally, the length of the probes will be about 20 to about 50 bases.The hybridization probes may be derived from the nucleotide sequence ofSEQ ID NO: 1, 3 or 5 or from genomic sequences including promoters,enhancer elements and introns of native sequence PRO201, PRO308 orPRO309. By way of example, a screening method will comprise isolatingthe coding region of the PRO201, PRO308 or PRO309 gene using the knownDNA sequence to synthesize a selected probe of about 40 bases.Hybridization probes may be labeled by a variety of labels, includingradionucleotides such as ³²P or ³⁵S, or enzymatic labels such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems. Labeled probes having a sequence complementary to that of thePRO201, PRO308 or PRO309 gene of the present invention can be used toscreen libraries of human cDNA, genomic DNA or mRNA to determine whichmembers of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

[0161] The probes may also be employed in PCR techniques to generate apool of sequences for identification of closely related PRO201, PRO308or PRO309 sequences.

[0162] Nucleotide sequences encoding a PRO201, PRO308 or PRO309 can alsobe used to construct hybridization probes for mapping the gene whichencodes that PRO201, PRO308 or PRO309 and for the genetic analysis ofindividuals with genetic disorders. The nucleotide sequences providedherein may be mapped to a chromosome and specific regions of achromosome using known techniques, such as in situ hybridization,linkage analysis against known chromosomal markers, and hybridizationscreening with libraries.

[0163] When the coding sequences for PRO201, PRO308 or PRO309 encode aprotein which binds to another protein (example, where the PRO201,PRO308 or PRO309 is a receptor), the PRO201, PRO308 or PRO309 can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO201, PRO308 or PRO309 can be used to isolate correlativeligand(s). Screening assays can be designed to find lead compounds thatmimic the biological activity of a native PRO201, PRO308 or PRO309 or areceptor for PRO201, PRO308 or PRO309. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

[0164] DNA30676 (SEQ ID NO:2) contains a single long open reading framewhich can encode a 576 amino acid protein, herein termed Nsp1 (novel SH2containing protein) (FIG. 1). Nsp1 (SEQ ID NO:1) is related to Sck andShc (See FIG. 8, SEQ ID NO:s 16 & 17) as determined by the indicatedN-terminal homology. Nsp1 (SEQ ID NO:1) also contains a proline-serinerich domain (P/S) in the middle of the protein that may function as anSH3 interaction domain. The C terminus of the protein has no obviouslyrelevant homology to any known mammalian proteins. Nsp1 (SEQ ID NO:1),Nsp2 (SEQ ID NO:3) and Nsp3 (SEQ ID NO:5) share an overall homologybetween 33 and 47%. Nsp3 (SEQ ID NO:5) has an SH2 domain and a potentialSH3 interaction domain, while Nsp2 (SEQ ID NO:3) lacks the SH2 domainbut does have a potential SH3 interaction domain. The absence of the SH2domain in Nsp2 (SEQ ID NO:3) is suggestive that this protein could actas a dominant negative regulator of the other two Nsps. The lack of anyapparent kinase or phosphatase domain suggests that they represent anovel family of adaptor proteins.

[0165] Adapter proteins are believed to play a significant roleintegrating multiple signaling cascades and in determining specificresponse to extracellular stimuli. Signals generated by growth factorssuch as EGF or IGF-1 through receptor tyrosine kinases or byextracellular matrix components acting through the integrin receptorscan induce cytoskeletal changes. Applicants have shown that the EGFreceptor coimmunoprecipitates with Nsp1 and is phosphorylated inresponse to EGF signaling. Thus, antagonists of Nsp1 would be expectedto be useful to inhibit cellular response attributed to stimulation bygrowth factors such as EGF or IGF-1 (e.g. tumorigenesis).

[0166] Several characteristics suggest that Nsp1 could play an importantrole in modulating the response to external stimuli. Nsp1 (SEQ ID NO:1)is phosphorylated in response to EGF stimulation and forms a complexthat includes the EGF receptor, PI3 kinase and Cas. The Nsp1/Cas complexalso responds to signaling through the fibronectin receptor. However,the stoichiometry of the interaction and the phosphorylation status ofthe components differs between the two stimuli. The implication is thatthat biological outcome in response to these extracellular signals couldbe quite distinct in the presence or absence of Nsp1. For example, FAKassociates with the SH3 region of Cas via a PXXP region at theC-terminus of FAK P(715)SRP—mouse nomenclature (Harte et al., J. Biol.Chem. 271: 13649-55 (1996). There are six PXXP signatures in Nsp1 (SEQID NO:1). This raises the possibility that Nsp1 could compete for theSH3 region on Cas and decrease the amount of Fak that is bound to Casand so alter Fak dependent events. The data also point to an EGFmediated decrease in the extent of phosphorylation of the Cas that isassociated with Nsp1 (SEQ ID NO:1). This complex then is likely to havea decrease in the number of proteins associated with the phosphorylatedtyrosines of the Cas and so lead to changes in downstream events. AsNsp1 (SEQ ID NO:1) expression is highest in fetal tissues this proteincould potentially have an important role in mediating the developmentalreadout of extracellular signals.

[0167] F. Amplification of Genes Encoding the PRO201, PRO308 or PRO309Polypeptides in Tumor Tissues and Cell Lines

[0168] The present invention is based in part on the identification andcharacterization of genes that are amplified in certain cancer cells.

[0169] The genome of prokaryotic and eukaryotic organisms is subjectedto two seemingly conflicting requirements. One is the preservation andpropagation of DNA as the genetic information in its original form, toguarantee stable inheritance through multiple generations. On the otherhand, cells or organisms must be able to adapt to lasting environmentalchanges. The adaptive mechanisms can include qualitative or quantitativemodifications of the genetic material. Qualitative modifications includeDNA mutations, in which coding sequences are altered resulting in astructurally and/or functionally different protein. Gene amplificationis a quantitative modification, whereby the actual number of completecoding sequence, i.e. a gene, increases, leading to an increased numberof available templates for transcription, an increased number oftranslatable transcripts, and, ultimately, to an increased abundance ofthe protein encoded by the amplified gene.

[0170] The phenomenon of gene amplification and its underlyingmechanisms have been investigated in vitro in several prokaryotic andeukaryotic culture systems. The best-characterized example of geneamplification involves the culture of eukaryotic cells in mediumcontaining variable concentrations of the cytotoxic drug methotrexate(MTX). MTX is a folic acid analogue and interferes with DNA synthesis byblocking the enzyme dihydrofolate reductase (DHFR). During the initialexposure to low concentrations of MTX most cells (>99.9%) will die. Asmall number of cells survive, and are capable of growing in increasingconcentrations of MTX by producing large amounts of DHFR-RNA andprotein. The basis of this overproduction is the amplification of thesingle DHFR gene.

[0171] The additional copies of the gene are found as extrachromosomalcopies in the form of small, supernumerary chromosomes (double minutes)or as integrated chromosomal copies.

[0172] Gene amplification is most commonly encountered in thedevelopment of resistance to cytotoxic drugs (antibiotics for bacteriaand chemotherapeutic agents for eukaryotic cells) and neoplastictransformation. Transformation of a eukaryotic cell as a spontaneousevent or due to a viral or chemical/environmental insult is typicallyassociated with changes in the genetic material of that cell. One of themost common genetic changes observed in human malignancies are mutationsof the p53 protein. p53 controls the transition of cells from thestationary (G1) to the replicative (S) phase and prevents thistransition in the presence of DNA damage. In other words, one of themain consequences of disabling p53 mutations is the accumulation andpropagation of DNA damage, i.e. genetic changes. Common types of geneticchanges in neoplastic cells are, in addition to point mutations,amplifications and gross, structural alterations, such astranslocations.

[0173] The amplification of DNA sequences may indicate specificfunctional requirement as illustrated in the DHFR experimental system.Therefore, the amplification of certain oncogenes in malignancies pointstoward a causative role of these genes in the process of malignanttransformation and maintenance of the transformed phenotype. Thishypothesis has gained support in recent studies. For example, the bcl-2protein was found to be amplified in certain types of non-Hodgkin'slymphoma. This protein inhibits apoptosis and leads to the progressiveaccumulation of neoplastic cells. Members of the gene family of growthfactor receptors have been found to be amplified in various types ofcancers suggesting that overexpression of these receptors may makeneoplastic cells less susceptible to limiting amounts of availablegrowth factor. Examples include the amplification of the androgenreceptor in recurrent prostate cancer during androgen deprivationtherapy and the amplification of the growth factor receptor homologueERB2 in breast cancer. Lastly, genes involved in intracellular signalingand control of cell cycle progression can undergo amplification duringmalignant transformation. This is illustrated by the amplification ofthe bcl-1 and ras genes in various epithelial and lymphoid neoplasms.

[0174] These earlier studies illustrate the feasibility of identifyingamplified DNA sequences in neoplasms, because this approach can identifygenes important for malignant transformation. The case of ERB2 alsodemonstrates the feasibility from a therapeutic standpoint, sincetransforming proteins may represent novel and specific targets for tumortherapy.

[0175] Several different techniques can be used to demonstrate amplifiedgenomic sequences. Classical cytogenetic analysis of chromosome spreadsprepared from cancer cells is adequate to identify gross structuralalterations, such as translocations, deletions and inversions. Amplifiedgenomic regions can only be visualized, if they involve large regionswith high copy numbers or are present as extrachromosomal material.While cytogenetics was the first technique to demonstrate the consistentassociation of specific chromosomal changes with particular neoplasms,it is inadequate for the identification and isolation of manageable DNAsequences. The more recently developed technique of comparative genomichybridization (CGH) has illustrated the widespread phenomenon of genomicamplification in neoplasms. Tumor and normal DNA are hybridizedsimultaneously onto metaphases of normal cells and the entire genome canbe screened by image analysis for DNA sequences that are present in thetumor at an increased frequency. (WO 93/18,186; Gray et al., RadiationRes. 137 275-289 [1994]). As a screening method, this type of analysishas revealed a large number of recurring amplicons (a stretch ofamplified DNA) in a variety of human neoplasms. Although CGH is moresensitive than classical cytogenetic analysis in identifying amplifiedstretches of DNA, it does not allow a rapid identification and isolationof coding sequences within the amplicon by standard molecular genetictechniques.

[0176] The most sensitive methods to detect gene amplification arepolymerase chain reaction (PCR)-based assays. These assays utilize verysmall amount of tumor DNA as starting material, are exquisitelysensitive, provide DNA that is amenable to further analysis, such assequencing and are suitable for high-volume throughput analysis.

[0177] The above-mentioned assays are not mutually exclusive, but arefrequently used in combination to identify amplifications in neoplasms.While cytogenetic analysis and CGH represent screening methods to surveythe entire genome for amplified regions, PCR-based assays are mostsuitable for the final identification of coding sequences, i.e. genes inamplified regions.

[0178] According to the present invention, such genes have beenidentified by quantitative PCR(S. Gelmini et al., Clin. Chem. 43:752[1997]), by comparing DNA from a variety of primary tumors, includingbreast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen,thymus, testis, ovary, uterus, etc. tumor, or tumor cell lines, withpooled DNA from healthy donors. Quantitative PCR was performed using aTaqMan instrument (ABI). Gene-specific primers and fluorogenic probeswere designed based upon the coding sequences of the DNAs.

[0179] Human lung carcinoma cell lines include A549 (SRC768), Calu-1(SRC769), Calu-6 (SRC770), H157 (SRC771), H441 (SRC772), H460 (SRC773),H522 (SRC832), H810 (SRC833), SKMES-1 (SRC774) and SW900 (SRC775), allavailable from ATCC. Primary human lung tumor cells usually derive fromadenocarcinomas, squamous cell carcinomas, large cell carcinomas,non-small cell carcinomas, small cell carcinomas, and broncho alveolarcarcinomas, and include, for example, SRC724 (squamous cell carcinomaabbreviated as “SqCCa”)(LTI), SRC725 (non-small cell carcinoma,abbreviated as “NSCCa”)(LT1a), SRC726 (adenocarcinoma, abbreviated as“AdenoCa”)(LT2), SRC727 (adenocarcinoma)(LT3), SRC728 (squamous cellcarcinoma)(LT4), SRC729 (adenocarcinoma)(LT6), SRC730 (adeno/squamouscell carcinoma)(LT7), SRCC731 (adenocarcinoma)(LT9), SRC732 (squamouscell carcinoma)(LT10), SRC733 (adenocarcinoma)(LT11), SRC734(adenocarcinoma)(LT12), SRC735 (broncho alveolar carcinoma, abbreviatedas “BAC”)(LT13), SRC736 (squamous cell carcinoma)(LT15), SRC737(squamous cell carcinoma)(LT16), SRC738 (squamous cell carcinoma)(LT17),SRC739 (squamous cell carcinoma)(LT18), SRC740 (squamous cellcarcinoma)(LT19), SRC741 (lung cell carcinoma, abbreviated as“LCCa”)(LT21), SRC811 (adenocarcinoma)(LT22).

[0180] Colon cancer cell lines include, for example, ATCC cell linesSW480 (adenocarcinoma, SRCC776), SW620 (lymph node metastasis of colonadenocarcinoma, SRC777), Colo320 (carcinoma, SRCC778), Colo205(carcinoma, SRC828), HCC2998 (carcinoma, SRC830), HT29 (adenocarcinoma,SRC779), HM7 (carcinoma, SRC780), KM12 (carcinoma, SRC831), CaWiDr(adenocarcinoma, SRC781), HCT15 (carcinoma, SRC829), HCT116 (carcinoma,SRC782), SKCO1 (adenocarcinoma, SRC783), SW403 (adenocarcinoma, SRC784),LS174T (carcinoma, SRC785), and HM7 (a high mucin producing variant ofATCC colon adenocarcinoma cell line LS 174T, obtained from Dr. RobertWarren, UCSF). Primary colon tumors include colon adenoocarcinomasdesignated CT1 (SRC751), CT2 (SRC742), CT3 (SRC743), CT4 (SRC752), CT5(SRC753), CT6 (SRC754), CT7 (SRC755), CT8 (SRC744), CT9 (SRC756), CT10(SRC745), CT11 (SRC757), CT12 (SRC746), CT14 (SRC747), CT15 (SRC748),CT16 (SRC749), CT17 (SRC750), CT18 (SRCC758).

[0181] Human breast carcinoma cell lines include, for example, HBL100(SRCC759), MB435s (SRCC760), T47D (SRCC761), MB468(SRCC762), MB175(SRCC763), MB361 (SRCC764), BT20 (SRCC765), MCF7 (SRCC766), SKBR3(SRCC767).

[0182] G. Tissue Distribution

[0183] The results of the gene amplification assays herein can beverified by further studies, such as, by determining mRNA expression invarious human tissues.

[0184] As noted before, gene amplification and/or gene expression invarious tissues may be measured by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad. Sci. USA, 77:5201-5205 [1980]), dot blotting (DNA analysis),or in situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.

[0185] Gene expression in various tissues, alternatively, may bemeasured by immunological methods, such as immunohistochemical stainingof tissue sections and assay of cell culture or body fluids, toquantitate directly the expression of gene product. Antibodies usefulfor immunohistochemical staining and/or assay of sample fluids may beeither monoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO213, PRO1330, PRO1449, PRO237, PRO324, PRO351, PRO362, PRO615,PRO531, PRO538, PRO3664, PRO618, PRO772 or PRO703 polypeptide or againsta synthetic peptide based on the DNA sequences provided herein oragainst exogenous sequence fused to sequence PRO213, PRO1330, PRO1449,PRO237, PRO324, PRO351, PRO362, PRO615, PRO531, PRO538, PRO3664, PRO618,PRO772 or PRO703 DNA and encoding a specific antibody epitope. Generaltechniques for generating antibodies, and special protocols for Northernblotting and in situ hybridization are provided hereinbelow.

[0186] H. Chromosome Mapping

[0187] If the amplification of a given gene is functionally relevant,then that gene should be amplified more than neighboring genomic regionswhich are not important for tumor survival. To test this, the gene canbe mapped to a particular chromosome, e.g. by radiation-hybrid analysis.The amplification level is then determined at the location identified,and at neighboring genomic region. Selective or preferentialamplification at the genomic region to which to gene has been mapped isconsistent with the possibility that the gene amplification observedpromotes tumor growth or survival. Chromosome mapping includes bothframework and epicenter mapping. For further details see e.g., Stewartet al., Genome Research 7, 422-433 (1997).

[0188] I. Antibody Binding Studies

[0189] The results of the gene amplification study can be furtherverified by antibody binding studies, in which the ability ofanti-PRO201, anti-PRO308 or anti-PRO309 to inhibit the expression of thePRO201, PRO308 or PRO309 polypeptides on tumor (cancer) cells is tested.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies, the preparation of whichwill be described hereinbelow.

[0190] Antibody binding studies may be carried out in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

[0191] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of target protein (encoded by agene amplified in a tumor cell) in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0192] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0193] For immunohistochemistry, the tumor sample may be fresh or frozenor may be embedded in paraffin and fixed with a preservative such asformalin, for example.

[0194] J. Cell-Based Tumor Assays

[0195] Cell-based assays and animal models for tumors (e.g cancers) canbe used to verify the findings of the gene amplification assay, andfurther understand the relationship between the genes identified hereinand the development and pathogenesis of neoplastic cell growth. The roleof gene products identified herein in the development and pathology oftumor or cancer can be tested by using primary tumor cells or cellslines that have been identified to amplify the genes herein. Such cellsinclude, for example, the breast, colon and lung cancer cells and celllines listed above.

[0196] In a different approach, cells of a cell type known to beinvolved in a particular tumor are transfected with the cDNAs herein,and the ability of these cDNAs to induce excessive growth is analyzed.Suitable cells include, for example, stable tumor cells lines such as,the B104-1-1 cell line (stable NIH-3T3 cell line transfected with theneu protooncogene) and ras-transfected NIH-3T3 cells, which can betransfected with the desired gene, and monitored for tumorogenic growth.Such transfected cell lines can then be used to test the ability ofpoly- or monoclonal antibodies or antibody compositions to inhibittumorogenic cell growth by exerting cytostatic or cytotoxic activity onthe growth of the transformed cells, or by mediating antibody-dependentcellular cytotoxicity (ADCC). Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of cancer.

[0197] In addition, primary cultures derived from tumors in transgenicanimals (as described below) can be used in the cell-based assaysherein, although stable cell lines are preferred. Techniques to derivecontinuous cell lines from transgenic animals are well known in the art(see, e.g. Small et al., Mol. Cell. Bio.l 5, 642-648 [1985]).

[0198] K. Animal Models

[0199] A variety of well known animal models can be used to furtherunderstand the role of the genes identified herein in the developmentand pathogenesis of tumors, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them particularly predictive of responses inhuman patients. Animal models of tumors and cancers (e.g. breast cancer,colon cancer, prostate cancer, lung cancer, etc.) include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing tumor cells into syngeneic miceusing standard techniques, e.g. subcutaneous injection, tail veininjection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, or orthopin implantation, e.g.colon cancer cells implanted in colonic tissue. (See, e.g. PCTpublication No. WO 97/33551, published Sep. 18, 1997).

[0200] Probably the most often used animal species in oncologicalstudies are immunodeficient mice and, in particular, nude mice. Theobservation that the nude mouse with hypo/aplasia could successfully actas a host for human tumor xenografts has lead to its widespread use forthis purpose. The autosomal recessive nu gene has been introduced into avery large number of distinct congenic strains of nude mouse, including,for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H, C57BL, C57, CBA,DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. Inaddition, a wide variety of other animals with inherited immunologicaldefects other than the nude mouse have been bred and used as recipientsof tumor xenografts. For further details see, e.g. The Nude Mouse inOncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc.,1991.

[0201] The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as, any of the above-listed tumor celllines, and, for example, the B 104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); a moderately well-differentiated grade IIhuman colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions, involvingfreezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer48: 689-696 [1983]).

[0202] Tumor cells can be introduced into animals, such as nude mice, bya variety of procedures. The subcutaneous (s.c.) space in mice is verysuitable for tumor implantation. Tumors can be transplanted s.c. assolid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue. Boven and Winograd (1991), supra.

[0203] Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogen wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al. PNAS USA 83: 9129-9133 (1986).

[0204] Similarly, animal models of colon cancer can be generated bypassaging colon cancer cells in animals, e.g. nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, by Wanget al., Cancer Research 54: 4726-4728 (1994) and Too et al., CancerResearch 55, 681-684 (1995). This model is based on the so-called“METAMOUSE” sold by AntiCancer, Inc. (San Diego, Calif.).

[0205] Tumors that arise in animals can be removed and cultured invitro. Cells from the in vitro cultures can then be passaged to animals.Such tumors can serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines.

[0206] For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 arechemically induced fibrosarcomas of BALB/c female mice (DeLeo et al., J.Exp. Med. 146: 720 [1977]), which provide a highly controllable modelsystem for studying the anti-tumor activities of various agents(Palladino et al., J. Immunol. 138: 4023-4032 [1987]). Briefly, tumorcells are propagated in vitro in cell culture. Prior to injection intothe animals, the cell lines are washed and suspended in buffer, at acell density of about 10×10⁶ to 10×10⁷ cells/ml. The animals are theninfected subcutaneously with 10 to 100 μl of the cell suspension,allowing one to three weeks for a tumor to appear.

[0207] In addition, the Lewis lung (3LL) carcinoma of mice, which is oneof the most thoroughly studied experimental tumors, can be used as aninvestigational tumor model. Efficacy in this tumor model has beencorrelated with beneficial effects in the treatment of human patientsdiagnosed with small cell carcinoma of the lung (SCCL). This tumor canbe introduced in normal mice upon injection of tumor fragments from anaffected mouse or of cells maintained in culture (Zupi et al., Br. J.Cancer 41: suppl. 4, 309 [1980]), and evidence indicates that tumors canbe started from injection of even a single cell and that a very highproportion of infected tumor cells survive. For further informationabout this tumor model see Zacharski, Haemostasis 16: 300-320 [1986]).

[0208] One way of evaluating the efficacy of a test compound in ananimal model is implanted tumor is to measure the size of the tumorbefore and after treatment. Traditionally, the size of implanted tumorshas been measured with a slide caliper in two or three dimensions. Themeasure limited to two dimensions does not accurately reflect the sizeof the tumor, therefore, it is usually converted into the correspondingvolume by using a mathematical formula. However, the measurement oftumor size is very inaccurate. The therapeutic effects of a drugcandidate can be better described as treatment-induced growth delay andspecific growth delay. Another important variable in the description oftumor growth is the tumor volume doubling time. Computer programs forthe calculation and description of tumor growth are also available, suchas the program reported by Rygaard and Spang-Thomsen, Proc. 6th Int.Workshop on Immune-Deficient Animals, Wu and Sheng eds., Basel, 1989,301. It is noted, however, that necrosis and inflammatory responsesfollowing treatment may actually result in an increase in tumor size, atleast initially. Therefore, these changes need to be carefullymonitored, by a combination of a morphometric method and flow cytometricanalysis.

[0209] Recombinant (transgenic) animal models can be engineered byintroducing the coding portion of the genes identified herein into thegenome of animals of interest, using standard techniques for producingtransgenic animals. A transgenic animal is one containing a “transgene”or genetic material integrated into the genome introduced into theanimal itself or an ancestor of the animal at a prenatal stage (e.g.,embryonic stage). Animals that can serve as a target for transgenicmanipulation include, without limitation, mice, rats, rabbits, guineapigs, sheep, goats, pigs, and non-human primates, e.g. baboons,chimpanzees and monkeys. Techniques known in the art to introduce atransgene into such animals include pronucleic microinjection (Hoppe andWanger, U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer intogerm lines (e.g., Van der Putten et al., Proc. Natl. Acad. Sci. USA 82:6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal., Cell 56: 313-321 [1989]); electroporation of embryos (Lo, Mol. Cel.Biol. 3: 1803-1814 [1983]); sperm-mediated gene transfer (Lavitrano eta., Cell 57: 717-73[1989]). For review, see, for example, U.S. Pat. No.4,736,866 and U.S. Pat. No. 4,870,009.

[0210] Typically, particular cells would be targeted for PRO201, PRO308or PRO309 transgene incorporation with tissue-specific enhancers.Transgenic animals that include a copy of a transgene encoding a PRO201,PRO308 or PRO309 introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding PRO201, PRO308 or PRO309. Such animals can beused as tester animals for reagents thought to confer protection from,for example, pathological conditions associated with its overexpression.In accordance with this facet of the invention, an animal is treatedwith the reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

[0211] For the purpose of the present invention, transgenic animalsinclude those that carry the transgene only in part of their cells(“mosaic animals”). The transgene can be integrated either as a singletransgene, or in concatamers, e.g., head-to-head or head-to-tailtandems. Selective introduction of a transgene into a particular celltype is also possible by following, for example, the technique of Laskoet al., Proc. Natl. Acad. Sci. USA 89: 6232-636 (1992).

[0212] The expression of the transgene in transgenic animals can bemonitored by standard techniques. For example, Southern blot analysis orPCR amplification can be used to verify the integration of thetransgene. The level of mRNA expression can then be analyzed usingtechniques such as in situ hybridization, Northern blot analysis, PCR,or immunocytochemistry. The animals are further examined for signs oftumor or cancer development.

[0213] Alternatively, “knock out” animals can be constructed which havea defective or altered gene encoding a PRO201, PRO308 or PRO309polypeptide identified herein, as a result of homologous recombinationbetween the endogenous gene encoding the polypeptide and altered genomicDNA encoding the same polypeptide introduced into an embryonic cell ofthe animal. For example, cDNA encoding a PRO201, PRO308 or PRO309polypeptide can be used to clone genomic DNA encoding that polypeptidein accordance with established techniques. A portion of the genomic DNAencoding a particular PRO201, PRO308 or PRO309 polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51: 503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69: 915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, by their ability to defend against certainpathological conditions and by their development of pathologicalconditions due to absence of the PRO201, PRO308 or PRO309 polypeptide.

[0214] The efficacy of antibodies specifically binding the polypeptidesidentified herein and other drug candidates, can be tested also in thetreatment of spontaneous animal tumors. A suitable target for suchstudies is the feline oral squamous cell carcinoma (SCC). Feline oralSCC is a highly invasive, malignant tumor that is the most common oralmalignancy of cats, accounting for over 60% of the oral tumors reportedin this species. It rarely metastasizes to distant sites, although thislow incidence of metastasis may merely be a reflection of the shortsurvival times for cats with this tumor. These tumors are usually notamenable to surgery, primarily because of the anatomy of the feline oralcavity. At present, there is no effective treatment for this tumor.Prior to entry into the study, each cat undergoes complete clinicalexamination, biopsy, and is scanned by computed tomography (CT). Catsdiagnosed with sublingual oral squamous cell tumors are excluded fromthe study. The tongue can become paralyzed as a result of such tumor,and even if the treatment kills the tumor, the animals may not be ableto feed themselves. Each cat is treated repeatedly, over a longer periodof time. Photographs of the tumors will be taken daily during thetreatment period, and at each subsequent recheck. After treatment, eachcat undergoes another CT scan. CT scans and thoracic radiograms areevaluated every 8 weeks thereafter. The data are evaluated fordifferences in survival, response and toxicity as compared to controlgroups. Positive response may require evidence of tumor regression,preferably with improvement of quality of life and/or increased lifespan.

[0215] In addition, other spontaneous animal tumors, such asfibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma ofdogs, cats, and baboons can also be tested. Of these mammaryadenocarcinoma in dogs and cats is a preferred model as its appearanceand behavior are very similar to those in humans. However, the use ofthis model is limited by the rare occurrence of this type of tumor inanimals.

[0216] L. Screening Assays for Drug Candidates

[0217] Screening assays for drug candidates are designed to identifycompounds that bind or complex with the polypeptides encoded by thegenes identified herein, or otherwise interfere with the interaction ofthe encoded polypeptides with other cellular proteins. Such screeningassays will include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Small molecules contemplated includesynthetic organic or inorganic compounds, including peptides, preferablysoluble peptides, (poly)peptide-immunoglobulin fusions, and, inparticular, antibodies including, without limitation, poly- andmonoclonal antibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

[0218] All assays are common in that they call for contacting the drugcandidate with a polypeptide encoded by a nucleic acid identified hereinunder conditions and for a time sufficient to allow these two componentsto interact.

[0219] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the polypeptide encoded by the gene identifiedherein or the drug candidate is immobilized on a solid phase, e.g. on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the polypeptide and drying. Alternatively, an immobilizedantibody, e.g. a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g. the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g. by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

[0220] If the candidate compound interacts with but does not bind to aparticular PRO201, PRO308 or PRO309 polypeptide encoded by a geneidentified herein, its interaction with that polypeptide can be assayedby methods well known for detecting protein-protein interactions. Suchassays include traditional approaches, such as, cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers [Fields and Song, Nature 340: 245-246 (1989); Chien etal., Proc. Natl. Acad. Sci. USA 88: 9578-9582 (1991)] as disclosed byChevray and Nathans [Proc. Natl. Acad. Sci. USA 89: 5789-5793 (1991)].Many transcriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, while the other one functioning as the transcription activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

[0221] Compounds that interfere with the interaction of a PRO201-,PRO308- or PRO309-encoding gene identified herein and other intra- orextracellular components can be tested as follows: usually a reactionmixture is prepared containing the product of the amplified gene and theintra- or extracellular component under conditions and for a timeallowing for the interaction and binding of the two products. To testthe ability of a test compound to inhibit binding, the reaction is runin the absence and in the presence of the test compound. In addition, aplacebo may be added to a third reaction mixture, to serve as positivecontrol. The binding (complex formation) between the test compound andthe intra- or extracellular component present in the mixture ismonitored as described hereinabove. The formation of a complex in thecontrol reaction(s) but not in the reaction mixture containing the testcompound indicates that the test compound interferes with theinteraction of the test compound and its reaction partner.

[0222] M. Compositions and Methods for the Treatment of Tumors

[0223] The compositions useful in the treatment of tumors associatedwith the amplification of the genes identified herein include, withoutlimitation, antibodies, small organic and inorganic molecules, peptides,phosphopeptides, antisense and ribozyme molecules, triple helixmolecules, etc. that inhibit the expression and/or activity of thetarget gene product.

[0224] For example, antisense RNA and RNA molecule act to directly blockthe translation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g. between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

[0225] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g. Rossi, Current Biology 4: 469-471 (1994), and PCTpublication No. WO 97/33551 (published Sep. 18, 1997).

[0226] Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g. PCTpublication No. WO 97/33551, supra.

[0227] These molecules can be identified by any or any combination ofthe screening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0228] N. Anti-PRO201, PRO308 or PRO309 Antibodies

[0229] The present invention further provides anti-PRO201, anti-PRO308or anti-PRO309 antibodies. Exemplary antibodies include polyclonal,monoclonal, humanized, bispecific, and heteroconjugate antibodies. Someof the more promising drug candidates according to the present inventionare antibodies and antibody fragments which may inhibit the productionor the gene product of the amplified genes identified herein and/orreduce the activity of the gene products.

[0230] 1. Polyclonal Antibodies

[0231] The anti-PRO201, anti-PRO308 or anti-PRO309 antibodies maycomprise polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan. Polyclonal antibodies canbe raised in a mammal, for example, by one or more injections of animmunizing agent and, if desired, an adjuvant. Typically, the immunizingagent and/or adjuvant will be injected in the mammal by multiplesubcutaneous or intraperitoneal injections. The immunizing agent mayinclude the PRO201, PRO308 or PRO309 polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

[0232] 2. Monoclonal Antibodies

[0233] The anti-PRO201, anti-PRO308 or anti-PRO309 antibodies may,alternatively, be monoclonal antibodies. Monoclonal antibodies may beprepared using hybridoma methods, such as those described by Kohler andMilstein, Nature, 256:495 (1975). In a hybridoma method, a mouse,hamster, or other appropriate host animal, is typically immunized withan immunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes may be immunized in vitro.

[0234] The immunizing agent will typically include the PRO201, PRO308 orPRO309 polypeptide or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes (“PBLs”) are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

[0235] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol, 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0236] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst PRO201, PRO308 or PRO309. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0237] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0238] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0239] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0240] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0241] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0242] 3. Humanized Antibodies

[0243] The anti-PRO201, PRO308 or PRO309 antibodies of the invention mayfurther comprise humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0244] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0245] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol Biol, 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boemer et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boemer et a., J. Immunol., 147(1):86-95 (1991)].

[0246] 4. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

[0247] The antibodies of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278.

[0248] The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such as way so asto convert it into its more active, cytotoxic form.

[0249] Enzymes that are useful in the method of this invention include,but are not limited to, glycosidase, glucose oxidase, human lysosyme,human glucuronidase, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases (e.g., carboxypeptidase G2and carboxypeptidase A) and cathepsins (such as cathepsins B and L),that are useful for converting peptide-containing prodrugs into freedrugs; D-alanylcarboxypeptidases, useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuramimidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin Vamidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

[0250] The enzymes of this invention can be covalently bound to theanti-PRO201, anti-PRO308 or anti-PRO309 antibodies by techniques wellknown in the art such as the use of the heterobifunctional cross-linkingagents discussed above. Alternatively, fusion proteins comprising atleast the antigen binding region of the antibody of the invention linkedto at least a functionally active portion of an enzyme of the inventioncan be constructed using recombinant DNA techniques well known in theart (see, e.g. Neuberger et al., Nature 312: 604-608 (1984)).

[0251] 5. Bispecific Antibodies

[0252] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the PRO201, PRO308 or PRO309, the other one is forany other antigen, and preferably for a cell-surface protein or receptoror receptor subunit.

[0253] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0254] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CHI) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0255] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0256] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229: 81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0257] Fab′ fragments may be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Me.d 175: 217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

[0258] Various technique for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers, Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992),wherein the leucine zipper peptides from the Fos and Jun proteins werelinked to the Fab′ portions of two different antibodies by gene fusion.The antibody homodimers were reduced at the hinge region to formmonomers and then re-oxidized to form the antibody heterodimers. Thismethod can also be utilized for the production of antibody homodimers.The “diabody” technology described by Hollinger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternativemechanism for making bispecific antibody fragments. The fragmentscomprise a heavy-chain variable domain (V_(H)) connected to alight-chain variable domain (V_(L)) by a linker which is too short toallow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152: 5368(1994).

[0259] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147: 60 (1991).

[0260] Exemplary bispecific antibodies may bind to two differentepitopes on a given “Pro” protein herein. Alternatively, an anti-“PRO”protein arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcγRI (CD64),FcγRII (CD32) and FcγRII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular “PRO” protein.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express a particular “PRO” polypeptide. These antibodiespossess a “PRO”-binding arm and an arm which binds a cytotoxic agent ora radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the “PRO” polypeptide and furtherbinds tissue factor (TF).

[0261] 6. Heteroconjugate Antibodies

[0262] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0263] 7. Effector Function Engineering

[0264] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance the effectiveness of theantibody in treating cancer, for example. For example cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3: 219-230 (1989).

[0265] 8. Immunoconjugates

[0266] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g. an enzymatically active toxin of bacterial, fungal,plant or animal origin, or fragments thereof, or a small moleculetoxin), or a radioactive isotope (i.e., a radioconjugate).

[0267] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active proteintoxins and fragments thereof which can be used include diphtheria Achain, nonbinding active fragments of diphtheria toxin, cholera toxin,botulinus toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin Achain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, saporin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes. Small molecule toxinsinclude, for example, calicheamicins, maytansinoids, palytoxin andCC1065. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

[0268] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0269] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g a radionucleotide).

[0270] 9. Immunoliposomes

[0271] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0272] Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989).

[0273] O. Uses for anti-PRO201, PRO308 or PRO309 Antibodies

[0274] The anti-PRO201, anti-PRO308 or anti-PRO309 antibodies of theinvention have various utilities. For example, anti-PRO201, anti-PRO308or anti-PRO309 antibodies may be used in diagnostic assays for PRO201,PRO308 or PRO309, e.g., detecting its expression in specific cells,tissues, or serum. Various diagnostic assay techniques known in the artmay be used, such as competitive binding assays, direct or indirectsandwich assays and immunoprecipitation assays conducted in eitherheterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: AManual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietymay be employed, including those methods described by Hunter et al.,Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Painet al., J. Immunol Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0275] Anti-PRO201, anti-PRO308 or anti-PRO309 antibodies also areuseful for the affinity purification of PRO201, PRO308 or PRO309 fromrecombinant cell culture or natural sources. In this process, theantibodies against PRO201, PRO308 or PRO309 are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the PRO201, PRO308 or PRO309 to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the PRO201,PRO308 or PRO309, which is bound to the immobilized antibody. Finally,the support is washed with another suitable solvent that will releasethe PRO201, PRO308 or PRO309 from the antibody.

[0276] P. PRO201, PRO308 and PRO309 Antagonists

[0277] Several approaches may be suitably employed to create theantagonist and agonist compounds of the present invention. Any approachwhere the antagonist molecule can be targeted to the interior of thecell, which interferes or prevents wild type PRO201, PRO308 or PRO309from normal operation is suitable. Additional properties of suchantagonist or agonist molecules are readily determinable by one ofordinary skill, such as size, charge and hydrophobicity suitable fortransmembrane transport.

[0278] Where mimics or other mammalian homologues of PRO201, PRO308 orPRO309 are to be identified or evaluated, the cells are exposed to thetest compound and compared to positive controls which are exposed onlyto PRO201, PRO308 or PRO309 and to negative controls which were notexposed to either the compound or the natural ligand. Where antagonistsor agonists of PRO201, PRO308 or PRO309 signal modulation are to beidentified or evaluated, the cells are exposed to the compound of theinvention in the presence of the natural ligand and compared to controlswhich are not exposed to the test compound.

[0279] Detection assays may by employed as a primary screen to evaluatethe phosphatase inhibition/enhancing activity of the antagonist/agonistcompounds of the invention. The assays may also be used to assess therelative potency of a compound by testing a range of concentrations, ina range from 100 mM to 1 pM, for example, and computing theconcentration at which the amount of phosphorylation or signaltransduction is reduced or increased by 50% (IC₅₀) compared to controls.

[0280] Assays can be performed to identify compounds that affectphosphorylation of PRO201, PRO308 or PRO309 substrates. Specifically,assays can be performed to identify compounds that increase thephosphorylation activity of PRO201, PRO308 or PRO309 or assays can beperformed to identify compounds that decrease the phosphorylation ofPRO201, PRO308 or PRO309 substrates. These assays can be performedeither on whole cells themselves or on cell extracts. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays, cell based assays,etc. Such assay formats are well known in the art.

[0281] The screening assays of the present invention are amenable tohigh-throughput screening of chemical libraries, and are particularlysuitable for identifying small molecule drug candidates.

[0282] (1) Antagonist and Agonist Molecules

[0283] To screen for antagonists and/or agonists of PRO201, PRO308 orPRO309 signaling, the assay mixture is incubated under conditionswhereby, but for the presence of the candidate pharmacological agent,PRO201, PRO308 or PRO309 induces intracellular signaling (for example,association of Nsp1 (SEQ ID NO:1) with the EGF receptor) with areference activity. The mixture components can be added in any orderthat provides for the requisite activity. Incubation may be performed atany temperature that facilitates optimal binding, typically betweenabout 4° C. and 40° C., more commonly between about 15° and 40° C.Incubation periods are likewise selected for optimal binding but alsominimized to facilitate rapid, high-throughput screening, and aretypically between about 0.1 and 10 hours, preferably less than 5 hours,more preferably less than 2 hours. After incubation, the effect of thecandidate pharmacological agent on the PRO201, PRO308 or PRO309signaling is determined in any convenient way. For cell-freebinding-type assays, a separation step is often used to separate boundand unbound components. Separation may, for example, be effected byprecipitation (e.g. TCA precipitation, immunoprecipitation, etc.),immobilization (e.g. on a solid substrate), followed by washing. Thebound protein is conveniently detected by taking advantage of adetectable label attached to it, e.g. by measuring radioactive emission,optical or electron density, or by indirect detection using, e.g.antibody conjugates.

[0284] Suitable molecules that affect the protein-protein interaction ofPRO201, PRO308 or PRO309 and its binding proteins include fragments ofthe latter or small molecules, e.g., peptidomimetics, which will preventinteraction and proper complex formation. Such small molecules, whichare usually less than 10 KD molecular weight, are preferable astherapeutics since they are more likely to be permeable to cells, areless susceptible to degradation by various cellular mechanisms, and arenot as apt to elicit an immune response as proteins. Small moleculesinclude but are not limited to synthetic organic or inorganic compounds.Many pharmaceutical companies have extensive libraries of suchmolecules, which can be conveniently screened by using the assays of thepresent invention. Non-limiting examples include proteins, peptides,glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosacchardies, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like.

[0285] A preferred technique for identifying molecules which bind toPRO201, PRO308 or PRO309 utilizes a chimeric substrate (e g.,epitope-tagged fused or fused immunoadhesin) attached to a solid phase,such as the well of an assay plate. The binding of the candidatemolecules, which are optionally labeled (e.g., radiolabeled), to theimmobilized receptor can be measured. Alternatively, competition forinteraction of Nspl with the binding proteins can be assayed. Furtheryet, molecules may be screened which affect the tumorigenicity ofPRO201, PRO308 or PRO309 in NIH3T3 cells in nude mice. In screening forantagonists and/or agonists, PRO201, PRO308 or PRO309 can be exposed toa PRO201, PRO308 or PRO309 substrate followed by the putative antagonistand/or agonist, or the PRO201, PRO308 or PRO309 binding protein andantagonist and/or agonist can be added simultaneously, and the abilityof the antagonist and/or agonist to block PRO201, PRO308 or PRO309activation can be evaluated.

[0286] (2) Detection Assays

[0287] The PRO201, —PRO308 or PRO309 polypeptides are useful in assaysfor identifying lead compounds for therapeutically active agents thatmodulate PRO201, PRO308 or PRO309 signaling. Specifically, leadcompounds that either prevent the formation of PRO201, PRO308 or PRO309signaling complexes or prevent or attenuate PRO201, PRO308 or PRO309modulated can be conveniently identified.

[0288] (a) Biochemical Detection Techniques

[0289] Biochemical analysis techniques can be evaluated by a variety oftechniques. One typical assay mixture which can be used with the presentinvention contains PRO201, PRO308 or PRO309 and a protein with whichPRO201, PRO308 or PRO309 is normally directly or indirectly associated(e.g. Cas), usually in an isolated, partially pure or pure form. One orboth of these components may be PRO201, PRO308 or PRO309 bound toanother peptide or polypeptide, which may, for example, provide orenhance protein-protein binding, improve stability under assayconditions, etc. In addition, one of the components usually comprises oris coupled to a detectable label. The label may provide for directdetection by measuring radioactivity, luminescence, optical or electrondensity, etc., or indirect detection such as an epitope tag, an enzyme,etc. The assay mixture can additionally comprise a candidatepharmacological agent, and optionally a variety of other components,such as salts, buffers, carrier proteins, e.g. albumin, detergents,protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.,which facilitate binding, increase stability, reduce non-specific orbackground interactions, or otherwise improve the efficiency orsensitivity of the assay.

[0290] The following detection methods may also be used in a cell-freesystem wherein cell lysate containing the signal transducing substratemolecule and PRO201, PRO308 or PRO309 is mixed with a compound of theinvention.

[0291] (i) Whole Cell Detection

[0292] A common technique involves incubating cells with vertebratePRO201, PRO308 or PRO309 and radiolabeled phosphate, lysing the cells,separating cellular protein components of the lysate using anSDS-polyacrylamide gel (SDS-PAGE) technique, in either one or twodimensions, and detecting the presence of phosphorylated proteins byexposing X-ray film. Detection can also be effected without usingradioactive labeling. In such a technique, the protein components (e.g.,separated by SDS-PAGE) are transferred to a nitrocellulose membranewhere the presence of phosphorylated tyrosine is detected using anantiphosphotyrosine antibody (anti-pTyr).

[0293] Alternatively, the anti-pTyr can be conjugated with an enzyme,such as horseradish peroxidase, and detected by subsequent addition of acolorimetric substrate for the enzyme. A further alternative involvesdetecting the anti-pTyr by reacting with a second antibody thatrecognizes the anti-pTyr, this second antibody being labeled with eithera radioactive moiety or an enzyme as previously described. Examples ofthese and similar techniques are described in Hansen et al.,Electrophoresis 14: 112-126 (1993); Campbell et al., J. Biol. Chem. 268:7427-7434 (1993); Donato et al., Cell Growth Diff. 3: 258-268 (1992);Katagiri et al., J. Immunol. 150: 585-593 (1993). Additionally, theanti-pTyr can be detected by labeling it with a radioactive substance,followed by scanning the labeled nitrocellulose to detect radioactivityor exposure of X-ray film.

[0294] (b) Biological Defection Techniques:

[0295] The ability of the antagonist/agonist compounds of the inventionto modulate the activity PRO201, PRO308 or PRO309, which itselfmodulates intracellular signaling, may also be measured by scoring formorphological or functional changes associated with ligand binding. Anyqualitative or quantitative technique known in the art may be appliedfor observing and measuring cellular processes which comes under thecontrol of PRO201, PRO308 or PRO309. For example, expression of Nsp1(SEQ ID NO:1) in NIH3T3 cells causes formation of morphologicallytransformation of the cells. The presence and or number of these focican be used as an indicator of biological efficacy of antagonists ofagonists of Nsp1 signaling.

[0296] The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosages for use in humans.The dosage of the compounds of the invention should lie within a rangeof circulating concentrations with little or no toxicity. The dosage mayvary within this range depending on the dosage form employed and theroute of administration.

[0297] (2) Antisense Nucleotides

[0298] Another preferred class of antagonists involves the use of genetherapy techniques, include the administration of antisense nucleotides.Applicable gene therapy techniques include single or multipleadministrations of therapeutically effective DNA or mRNA. Antisense RNAsand DNAs can be used as therapeutic agents for blocking the expressionof certain genes in vivo. Short antisense oligonucleotides can beimported into cells where they act as inhibitors, despite their lowintracellular concentrations caused by restricted uptake by the cellmembrane, Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146(1986). The oligonucleotides can be modified to enhance their uptake,e.g., by substituting their negatively charged phophodiester groups byuncharged groups.

[0299] There are a variety of techniques known for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, ex vivo, or invivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection, Dzau et al., Trends Biotech. 11:205-210 (1993). In some situations it is desirable to provide thenucleic acid source with an agent that targets the target cells, such asan antibody specific for a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.,capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling, andproteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432(1987); Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990).For a review of known gene marking and gene therapy protocols, seeAnderson et al., Science 256: 808-813 (1992).

[0300] In one embodiment, PRO201, PRO308 or PRO309 antagonist and/oragonist molecules may be used to bind endogenous ligand in the cell,thereby causing the cell to be unresponsive to PRO201, PRO308 or PRO309wild type, especially when the levels of PRO201, PRO308 or PRO309 in thecell exceed normal physiological levels. Also, it may be beneficial tobind endogenous PRO201, PRO308 or PRO309 substrates or complexing agentsthat are activating undesired cellular responses (such as proliferationof tumor cells).

[0301] In a further embodiment of the invention, PRO201, PRO308 orPRO309 expression may be reduced by providing PRO201-, PRO308- orPRO309-expressing cells with an amount of PRO201, PRO308 or PRO309antisense RNA or DNA effective to reduce expression of the PRO201,PRO308 or PRO309 protein.

[0302] In a further embodiment of the invention, the expression ofbinding partners of PRO201, PRO308 or PRO309 may be reduced by providingPRO201, PRO308 or PRO309 expressing cells with an amount of antisenseRNA or DNA effective for reduced expression of the binding partners ofPRO201, PRO308 or PRO309.

[0303] Q. Diagnostic Uses

[0304] Another use of the compounds of the invention (e.g., PRO201,PRO308 or PRO309, PRO20 I—, PRO308- or PRO309-variants and anti-PRO201,anti-PRO308 or anti-PRO309 antibodies) described herein is to helpdiagnose whether a disorder is driven, to some extent, by PRO201, PRO308or PRO309 modulated signaling.

[0305] A diagnostic assay to determine whether a particular disorder isdriven by PRO201, PRO308 or PRO309 signaling, can be carried out usingthe following steps: (1) culturing test cells or tissues; (2)administering a compound which can inhibit Nsp1, Nsp2 or Nsp3 modulatedsignaling; and (3) measuring the degree of phosphorylation on thePRO201, PRO308 or PRO309 substrate in cell lysates or PRO201, PRO308 orPRO309 mediated phenotypic effects in the test cells. The steps can becarried out using standard techniques in light of the presentdisclosure. For example, standard techniques can be used to isolatecells or tissues and culturing or in vivo.

[0306] Compounds of varying degree of selectivity are useful fordiagnosing the role of PRO201, PRO308 or PRO309. For example, compoundswhich inhibit PRO201, PRO308 or PRO309 in addition to another form ofadaptor molecule can be used as an initial test compound to determine ifone of several adaptor molecules drive the disorder. The selectivecompounds can then be used to further eliminate the possible role of theother adaptor proteins in driving the disorder. Test compounds should bemore potent in inhibiting intracellular signaling activity than inexerting a cytotoxic effect (e.g., an IC₅₀/LD₅₀ of greater than one).The IC₅₀ and LD₅₀ can be measured by standard techniques, such as an MTTassay, or by measuring the amount of LDH released. The degree ofIC₅₀/LD₅₀ of a compound should be taken into account in evaluating thediagnostic assay. Generally, the larger the ratio the more relative theinformation. Appropriate controls take into account the possiblecytotoxic effect of a compound of a compound, such as treating cells notassociated with a cell proliferative disorder (e.g., control cells) witha test compound, can also be used as part of the diagnostic assay. Thediagnostic methods of the invention involve the screening for agentsthat modulate the effects of fused upon hedgehog signaling. Exemplarydetection techniques include radioactive labeling andimmunoprecipitating (U.S. Pat. No. 5,385,915).

[0307] While cell surface proteins, such as growth receptorsoverexpressed in certain tumors are excellent targets for drugcandidates or tumor (e.g. cancer) treatment, the same proteins alongwith secreted proteins encoded by the genes amplified in tumor cellsfind additional use in the diagnosis and prognosis of tumors. Forexample, antibodies directed against the proteins products of genesamplified in tumor cells can be used as tumor diagnostics orprognostics.

[0308] For example, antibodies, including antibody fragments, can beused to qualitatively or quantitatively detect the expression ofproteins encoded by the amplified genes (“marker gene products”). Theantibody preferably is equipped with a detectable, e.g. fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the amplified gene encodes a cell surfaceprotein, e.g. a growth factor. Such binding assays are performedessentially as described in section 5 above.

[0309] In situ detection of antibody binding to the marker gene productscan be performed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

[0310] R. Pharmaceutical Compositions

[0311] Antibodies specifically binding the product of an amplified geneidentified herein, as well as other molecules identified by thescreening assays disclosed hereinbefore, can be administered for thetreatment of tumors, including cancers, in the form of pharmaceuticalcompositions.

[0312] If the protein encoded by the amplified gene is intracellular andwhole antibodies are used as inhibitors, internalizing antibodies arepreferred. However, lipofections or liposomes can also be used todeliver the antibody, or an antibody fragment, into cells. Whereantibody fragments are used, the smallest inhibitory fragment whichspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable region sequences of anantibody, peptide molecules can be designed which retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology (see, e.g.Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 [1993]).

[0313] Therapeutic formulations of the antibody are prepared for storageby mixing the antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0314] Non-antibody compounds identified by the screening assays of thepresent invention can be formulated in an analogous manner, usingstandard techniques well known in the art.

[0315] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

[0316] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0317] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0318] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g. films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0319] S. Methods of Treatment

[0320] It is contemplated that the antibodies and other anti-tumorcompounds of the present invention may be used to treat variousconditions, including those characterized by overexpression and/oractivation of the amplified genes identified herein. Exemplaryconditions or disorders to be treated with such antibodies and othercompounds, including, but not limited to, small organic and inorganicmolecules, peptides, antisense molecules, etc. include benign ormalignant tumors (e.g. renal, liver, kidney, bladder, breast, gastric,ovarian, colorectal, prostate, pancreatic, ling, vulval, thyroid,hepatic carcinomas; sarcomas; glioblastomas; and various head and necktumors); leukemias and lymphoid malignancies; other disorders such asneuronal, glial, astrocytal, hypothalamic and other glandular,macrophagal, epithelial, stromal and blastocoelic disorders; andinflammatory, angiogenic and immunologic disorders.

[0321] The anti-tumor agents of the present invention, e.g. antibodies,are administered to a mammal, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. Intravenousadministration of the antibody is preferred.

[0322] Other therapeutic regimens may be combined with theadministration of the anti-cancer agents, e.g. antibodies of the instantinvention. For example, the patient to be treated with such anti-canceragents may also receive radiation therapy. Alternatively, or inaddition, a chemotherapeutic agent may be administered to the patient.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). Thechemotherapeutic agent may precede, or follow administration of theanti-tumor agent, e.g. antibody, or may be given simultaneouslytherewith. The antibody may be combined with an anti-oestrogen compoundsuch as tamoxifen or an anti-progesterone such as onapristone (see, EP616812) in dosages known for such molecules.

[0323] It may be desirable to also administer antibodies against othertumor associated antigens, such as antibodies which bind to the ErbB2,EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF).Alternatively, or in addition, two or more antibodies binding the sameor two or more different antigens disclosed herein may beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient. In a preferredembodiment, the antibodies herein are co-administered with a growthinhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by an antibody of the present invention.However, simultaneous administration or administration of the antibodyof the present invention first is also contemplated. Suitable dosagesfor the growth inhibitory agent are those presently used and may belowered due to the combined action (synergy) of the growth inhibitoryagent and the antibody herein.

[0324] For the prevention or treatment of disease, the appropriatedosage of an anti-tumor agent, e.g. an antibody herein will depend onthe type of disease to be treated, as defined above, the severity andcourse of the disease, whether the agent is administered for preventiveor therapeutic purposes, previous therapy, the patient's clinicalhistory and response to the agent, and the discretion of the attendingphysician. The agent is suitably administered to the patient at one timeor over a series of treatments.

[0325] For example, depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g 0.1-20 mg/kg) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

[0326] T. Articles of Manufacture

[0327] In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing ortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The active agentin the composition is usually an anti-tumor agent capable of interferingwith the activity of a gene product identified herein, e.g. an antibody.The label on, or associated with, the container indicates that thecomposition is used for diagnosing or treating the condition of choice.The article of manufacture may further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

[0328] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0329] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0330] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Rockville, Md.

Example 1 Isolation of cDNA Clones Encoding Human PRO201, PRO308 orPRO309

[0331] An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified (1328938, DNA28710, FIG. 4A)(SEQ ID NO:13) which was in afetal pancreas library which shared significant identity which theadaptor protein Shc. A full length cDNA corresponding to the isolatedEST was cloned from a human fetal kidney library using an in vivocloning technique (DNA30676, Nsp1)(SEQ ID NO:1) in pRK5. There is asingle long open reading frame which encodes a 576 amino acid protein.Nsp1 (SEQ ID NO:1) also is related to Sck and Shc (FIG. 8)(SEQ ID NO:s17 & 16, respectively, which is apparent in the SH2 region that appearsat the N-terminus of Nsp1 (SEQ ID NO:1). Nsp1 (SEQ ID NO:1) alsocontains a proline-serine rich domain (PS) in the middle of the proteinthat may function as an SH3 interaction domain. The C-terminus of Nsp1has no significant identity to any known mammalian proteins. ThisC-terminal sequence was then used to rescreen the EST database, whereinwas found two additional fragments (104191)(DNA38653)(FIG. 4B)(SEQ IDNO:14) and (1651811)(DNA38654)(FIG. 4C)(SEQ ID NO:15). From thesesequences were constructed cloning and enrichment primers, and thecorresponding full length sequences were isolated for Nsp2 (SEQ ID NO:3)and Nsp3 (SEQ ID NO:5), respectively, using an in vivo cloning techniquefrom a human placenta library in pRK5. The probes used for the cloningof the full length sequences were the following: Nsp1 (SEQ ID NO: 1):Nsp1 (SEQ ID NO:1): Cloning: ACTGAGGCCTGTTGAAAGTGCAGAGCTCAG (SEQ IDNO:7) Enrichment Primer: GCTGAAGAAGAGCTTCAG (SEQ ID NO:8) Nsp2 (SEQ IDNO:3): Cloning: CAATGCCGATGGCCATTGTGTTGTGTCTTTCAATTATGTCCAGGCGCA (SEQ IDNO:9) Enrichment Primer: ATCCCAGAATGTCCACTG (SEQ ID NO:10) Nsp3 (SEQ IDNO:5): Cloning: GGCCAGCATGATGGACATGGTGTGGAACCTTTCCAGCAGGTCTAGGCGTA (SEQID NO:11) Enrichment Primer: GGTGCAGCCCAGGATGTC (SEQ ID NO:12)

[0332] The three proteins (Nsp1, Nsp2, Nsp3)(SEQ ID NO:s 1, 3 & 5) sharean overall identity of between 33% and 47% (FIG. 6B). Nsp3 has an SH2domain and a potential SH3 interaction domain (PS region). Nsp2 lacksthe SH2 domain but does have a potential SH3 interaction domain. Theabsence of the SH2 domain in Nsp2 raises the possibility that thisprotein could act as a dominant negative regulator of the other twoNsps. All three proteins lack apparent kinase or phosphatase domains.

[0333] cDNA clones Nsp1, Nsp2 and Nsp3 (SEQ ID NO:s 2, 4 & 6) weresequenced in their entirety. The entire nucleotide sequence of DNA30676,DNA40575 and DNA61601 is shown in FIG. 1 (SEQ ID NO: 2), FIG. 2 (SEQ IDNO: 4) and FIG. 3 (SEQ ID NO: 6), respectively. Clones DNA30676-1223,DNA40575-1223 and DNA61601-1223 have been deposited with ATCC and areassigned ATCC deposit numbers 209567, 209565 and 209713. Moreover,related clones DNA40556-1223 and DNA40554-1223 have also been depositedwith the ATCC and are assigned ATCC deposit numbers 209566 and 2096564.

Example 2 Northern Blot Analysis

[0334] Expression of PRO201, PRO308 and PRO309 mRNA in human tissues wasexamined by northern blot analysis. Human RNA blots were hybridized toan ³²P-endlabelled DNA probe complementary to the nucleotide encodingamino acids: (a) 90-102 in DNA30676; (b) 270-284 in DNA40575 or (c)475-491 in DNA61601. Endocrine and fetal 11 (Clontech) were hybridizedin ExpressHyb® hybridization solution (Clontech) in accordance with themanufacturer's instructions. Blots were incubated with the probes inhybridization buffer (5×SSPE; 2× Denhardt's solution; 100 mg/mLdenatured sheared salmon sperm DNA; 50% formamide; 2% SDS) for 16 hoursat 42° C. The blots were washed several times in 2×SSC; 0.05% SDS for 1hour at room temperature, followed by a 30 minute wash in 0.1×SSC; 0.1%SDS at 50° C. The blots were developed after overnight exposure byphosphorimager analysis (Fuji).

[0335] As shown in FIG. 9A, significant expression of Nsp1 (SEQ ID NO:2)was only detected in human fetal liver and may be expressed in otherfetal tissues (e.g., fetal kidney). This pattern of expression suggestsa role for Nsp1 in coordinating signaling pathways important for fetaldevelopment. In contrast, Nsp2 (SEQ ID NO:4) and Nsp3 (SEQ ID NO:6) weremore widely expressed in many tissues. In hematopoietic tissues, twoNsp2 transcripts (3.8 Kb and 3.2 Kb) were detected (FIG. 9B).

Example 3 EGF, Insulin and Fibronectin induced Nsp1 Phosphorylation andComplex Formation with p130^(Cas)

[0336] As Nsp1 has three potentially phosphorylatable tyrosines, a studywas undertaken to determine whether Nsp1 could be phosphorylated inresponse to a variety of extracellular stimuli. Treatment with EGFinduced a rapid tyrosine phosphorylation of Nsp1 (SEQ ID NO:1) whichoccurred in 2 minutes or less (FIG. 10A). Nsp1 (SEQ ID NO:1) is alsophosphorylated in response to insulin, IGF-1 and heregulin (not shown).In contrast, fibronectin (FN) stimulated only weak Nsp1 phosphorylation(FIG. 10C).

[0337] In order to trace the pathway(s) impacted by Nsp1 (SEQ ID NO: 1).Applicants have identified proteins associated with Nsp1 (SEQ ID NO:1)in vivo by way of co-immunoprecipitation experiments. Treatment with EGFlead to an association between Nsp1 (SEQ ID NO: 1) and a tyrosinephosphorylated protein with a molecular mass of approximately 170 kD.This protein is rapidly tyrosine phosphorylated in response to EGF andcan be detected with a mAB directed against the EGF receptor. Further,Nsp1 (SEQ ID NO:1) can be detected by western blotting followingimmunoprecipitation of the EGF receptors (FIG. 10B). There is residualNsp1/EGF receptor interaction prior to EGF treatment, but the extent ofthe interaction significantly increases following exposure to EGF.

[0338] The coimmunoprecipitation experiments also revealed that Nsp1interacts with a 130 kD protein (p 130). In serum starved cells p130 wasphosphorylated to a moderate level (FIG. 10A), whereas loss of cellattachment lead to a complete p130 dephosphorylation (FIG. 10C). Bywestern blotting analysis this p130 was found to the adaptor proteinp130^(Cas). In FIG. 10A, anti-(P)Tyr antibody detected two bands atapproximately 130 kD, while anti-Cas antibody recognizes only the bottomband. We have not yet identified the upper band. Cas was originallyfound as a hyper-phosphorylated protein following induced expression ofviral Crk (v-Crk) [Sakai et al., EMBO J. 13: 3748-56 (1994)] and isphosphorylated in response to integrin interaction with extracellularmatrix as well as a number of other stimuli. Chen et al., J. Biol. Chem.272: 27401-10 (1997); Casamassima & Rozengurt, J. Biol. Chem. 272;9363-70 (1997); Nojima et al., J. Biol. Chem. 270: 15398-402 (1995). Casdirectly interacts with focal adhesion kinase (FAK)[Polte & Hanks, Proc.Natl. Acad. Sci. USA 92: 10678-82 (1995)] and appears to be a criticalcomponent by which extracellular events influence cell motilitymorphology and survival. Daniel & Reynolds, Mol. & Cell. Biol 15:4819-24 (1995); Mo & Reynolds, Cancer Res. 56: 2633-40 (1996); Nakamotoet al., Mol. Cell. Biol. 17: 3884-97(1997).

[0339] The phosphorylation status of Nsp1 (SEQ ID NO:1) and Cas and therelative amount of Cas associated with Nsp1 (SEQ ID NO:1) is dependenton the signaling through either the EGF or integrin receptors. EGFincreases Nsp1 phosphorylation but dephosphorylation of both total (datanot shown) and Nsp1 (SEQ ID NO:1) associated Cas (FIG. 10A). There isalso an increase in the amount of Cas associated with Nsp1 after EGFtreatment (FIG. 10A). In contrast, fibronectin had only a small effecton Nsp1 (SEQ ID NO:1) phosphorylation but increased the phosphorylationof Cas that is associated with Nsp1 and at the same time lead to atransient decrease in the amount of Cas that is associated with Nsp1(FIG. 10C). An increase in Cas phosphorylation in response to integrinshas been previously reported. Nojima et al., J. Biol. Chem. 270:15398402 (1995). This decrease in the Nsp1/Cas complex reached a nadirat approximately 30 minutes and then returned toward baseline conditionsat around 4 hours.

[0340] In FIG. 11 it is demonstrated that insulin stimulated Nsp1phosphorylation peaked at 2 hours, and then decreased after 14.5 hours.The same blot was reprobed with anti-FLAG antibody to show the equalloading. In FIG. 11 it is demonstrated that IGF-I also stimulates thephosphorylation of Nsp1 although the level of phosphorylation inresponse to IGF-1 is less than that seen in response to insulin.

[0341] IGF-1 Results:

[0342] In the absence of insulin (FIG. 13) or EGF (not shown) Nsp1 isassociated with Cas. The phosphorylation of Cas was observed to decreaseafter insulin treatment. This is indicated both in FIG. 12 and FIG. 13A.The membrane of the tested samples in FIG. 13A was stripped and reprobedwith anti-p130^(Cas) antibody to also demonstrate that the amount of Casassociated with Nsp1 decreases following insulin treatment.

[0343] Materials and Methods:

[0344] EGF/Fibronectin:

[0345] Transfected and serum starved COS (A,B) were either treated with25 ng/ml EGF for the times indicated or left untreated. Transfected andserum starved 293 cells were either attached to plastic (on dish), heldin suspension (off dish) or replated onto 10 mg/ml FN-coated dishes forthe times indicated FIG. 10C(C). In FIGS. 10A and 10C anti-flagimmunoprecipitates were blotted with anti-flag, anti-(P)Tyr or anti-Casantibodies as indicated. In FIG. 10B, anti-EGF receptor (CalBiochem)immunoprecipitates were blotted with anti-flag or anti-(P)Tyr antibodiesas indicated. Transfected cells were lysed in coimmunoprecipitationassay (ColPA) buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100,2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mMorthovanadate) containing freshly added protease inhibitors (1 mM AEBSF,10 mM leupeptin, 2 mg/ml aprotinin, 1 mM pepstatin). Anti-flag (Kodak,IBI immunoprecipitates and the associated proteins were visualized byanti-(P)Tyr antibody PY20 (Transduction Lab). The same blots werestriped and reblotted with anti-p130^(Cas) (Transduction Lab) oranti-flag antibody and detected with the ECL system (Pierce). The Flapepitope (DYKDDDDK)(SEQ ID NO:19) was added in frame to the N-terminus ofthe Nsp1 cDNA construct using in vitro mutagenesis to createpRK.Nsp1.FLAG.

[0346] Insulin:

[0347] The FLAG epitope (DYKDDDDK)(SEQ ID NO: 19) was inserted into theN-terminus of the Nsp1 cDNA construct using a standard in vitromutagenesis to create pRK.Nsp.FLAG. CHO cells overexpressing insulinreceptor (CHO-IR) were cultured in F12-DMEM containing 10% serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.Liposome-mediated transfection methods using DOSPER (BoehringerMannheim) or superfect (Qiagen) were carried out on CHO cells inaccordance with the manufacturers instructions. CHO-IR cells weretransiently transfected with either the empty vector pRK or withpRK.Nsp1.FLAG and serum starved for 16 hours. Cells were treated with orwithout 100 nM insulin for different times and then lysed on ice for onehour in 1 ml of immunoprecipitation assay (IPA) buffer (10 mM Tris, pH7.5, 150 mM CaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EGTA,10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM orthovanadate)containing fresh added protease inhibitors (1 mM AEBSF, 10 μM leupeptin,2 μg/ml aprotinin, 1 μM pepstatin). Samples were immunoprecipitated withan anti-FLAG affinity gel (IBI, Kodak). Following SDS-polyacrylamide gelelectrophoresis, proteins were transferred onto nitrocellulose membrane(Novex), western blotted with the anti-phosphotyrosine antibody PY-20(Transduction Lab) or anti-FLAG antibodies and detected with the ECLsystem (Pierce).

[0348] IGF-1

[0349] pRK or pRK.Nsp1.FLAG transfected with CHO-IR or CHO-IGFIR (IGF-1receptor) cells were serum starved, treated with 100 nM insulin or 100ng/ml IGF-1 and lysed in coimmunoprecipitation assay (CoIPA) buffer (20mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10 mM sodiumpyrophosphate, 10 mM sodium fluoride, 2 mM orthovanadate) containingprotease inhibitors. Samples were immunoprecipitated with anti-FLAG oranti-p130^(Cas) and Western blotted with the anti-phosphotyrosineantibody PY-20 or anti-p130Cas (Santa Cruz Biotechnology).

Example 4 Mapping of Phosphorylated Tyrosine Residues in Nsp1

[0350] In order to map the phosphorylated residues in Nsp1, Applicantshave independently changed each of the three tyrosine in Nsp1 (SEQ IDNO:1) to phenylalanine. Transfected cells were then stimulated with EGFand the Nsp1 (SEQ ID NO:1) immunoprecipitated. In all three cases thenon-phosphorylated Nspl (SEQ ID NO:1) immunoprecipitated fromnon-stimulated cells was associated with both Cas and the EGF receptor.These results demonstrate that the amino acid changes were not grosslydeleterious to the overall protein structure. While mutant Nsp1_(Y61)F(SEQ ID NO:21) was phosphorylated normally in response to EGF,phosphorylation of Nsp1_(Y95F) (SEQ ID NO:22) was not detected andNsp1_(Y231F) (SEQ ID NO:23) was weakly phosphorylated. This datasuggests that there is first a phosphorylation of Y95 followed by thephosphorylation of Y231. Y16 may or may not be phosphorylated, but isnot required for phosphorylation of either Y95 or Y231. Further, as theamount of EGF receptor coimmunoprecipitated with Nsp1 (SEQ ID NO:1) isincreased by receptor phosphorylation but largely independent of Nsp1(SEQ ID NO:1) phosphorylation, it would appear that Nsp1 (SEQ ID NO:1)association with Cas is independent of both Nsp1 (SEQ ID NO:1) and Casphosphorylation status (FIG. 10), this interaction may be mediatedthrough the SH3 domain of Cas and SH3 interaction domain of Nsp1 (SEQ IDNO: 1).

[0351] Materials and Methods:

[0352] All three tyrosine residues in Nsp1 (SEQ ID NO: 1) were changedto phenylalanine using a standard in vitro mutagenesis technique.Mutants (Y16F, Y95F and Y231F) (SEQ ID NO:s 21-23, respectively) andwild type Nsp1 (SEQ ID NO:1) DNAs were transfected into COS cells andtreated with 25 ng/ml of EGF for 10 min. or left untreated. Cell lysateswere immunoprecipitated with the anti-flag antibody and Western blottedwith either the anti-P(Tyr) antibody, the anti-p130^(Cas) antibody orthe anti-flag antibody.

Example 5 Transformation and Tumorigenicity in Nude Mice

[0353] Introduction:

[0354] Since Cas has been implicated in c-src mediated events [Sakai etal., EMBO J. 13: 3748-56 (1994); Sakai et al., Oncogene 14: 1419-26(1997)], Applicants examined the effect of Nsp1 (SEQ ID NO:1) in anNIH3T3 transformation assay.

[0355] The NIH3T3 is a cell line which normally grow in monolayer evenwhen the cells are overconfluent. They may be used to determine whetheror not a candidate gene has the potential for oncogenicity when thecandidate is transfected via retroviral mediated infection in vectorMSCV. The transfected cells are allowed to generate into foci, pickedand cultured to 10 million cells and injected into nude mice. It thetransfected gene is oncogenic, it will grow on uninhibited by thedeficient immune system of the nude mouse and form a tumor. See Winogradet al., In Vivo 1 (1): 1-13 (1987).

[0356] Discussion and Results:

[0357] More than one hundred foci of morphologically transformed cellswere observed on one 100 mm plate of NIH3T3 cells following transfectionwith a retrovirus expressing Nsp1 and G418 selection, but none appearedin control (neo) vector. The transformed cells (FIG. 14B) were morerounded and compacted in comparison to the normal elongated fibroblastshape of the control transfected NIH3T3 cells (FIG. 14A). To investigatewhether the transformed Nsp1 expressing cells were also tumorigenic,three independent foci were picked and expanded to generateNIH3T3-MSCV.Nsp1-.sub1, -.sub2 and -.sub3. Controls consisted of celllines expressing neo only (NIH3T3-neo) and a pool of transfected cellsthat expressed lower levels of Nsp1 (SEQ ID NO:2) but was nottransformed (NIH3T3-Nsp1.non-trans). The Nsp1 (SEQ ID NO:2) expressing,non-transformed cells were derived by infecting NIH3T3 cells with theNsp1 (SEQ ID NO:2) expressing retroviral vector. These bulk cultureswere selected for neomycin resistance, but were not allowed to proceedthrough the postconfluent growth that selects for foci formation.

[0358] Each cell line was injected into five mice. No tumor growth wasobserved in any mice injected with neo control cells orNIH3T3-Nsp1.non-trans. cells. All five mice in each group injected withNIH3T3-MSCV.Nsp1-.sub1, -.sub2, -.sub3 grew obvious tumors within threeweeks. Histological analysis indicated that the tumors consisted oflarge, irregular, moderately anaplastic epithelioid cells with a highmitotic index (FIG. 14C). There was no evidence of metastasis.

[0359] The tumors which formed were well circumscribed, locallyexpansile masses composed primarily of larger, irregular, moderatelyanaplastic epitheloid cells with a high mitotic index, interspersed,peripherally by small areas of spindle-cell proliferation. (FIG. 14C).

[0360] Materials and Methods:

[0361] pRK.Nsp1.FLAG plasmid was digested with EcoRI and Sal I. The Nsp1cDNA fragment including the FLAG epitope was purified and subcloned intothe EcoRI and Xho I sites of the retroviral vector MSCVneo resulting inMSCVneo.Nsp1.FLAG. Mouse embryonic fibroblast cells (ATCC) andretroviral producer BOSC 23 cells were maintained in DMEM with 10% fetalbovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/mlstreptomycin.

[0362] The retroviral vectors MSCVneo and MSCVneo.Nspl.FLAG weretransfected into BOSC 23 cells using calcium phosphate-mediatedtransfection. The 72-hour supernatant was used to infect NIH3T3 cellsplated on a 6-well plate. Infected cells were selected in 400 μg/ml G418(Gibco) and pooled to generate NIH3T3-MSCV and -MSCV.Nsp1 cell lines.NIH3T3-MSCV and -MSCV.Nsp1 cells were grown until confluent for 4 dayswith a medium change once, split at a one to five ratio and grown untilconfluent for another 4 days. More than one hundred foci ofmorphologically transformed cells were observed on one 100 mm plate ofNIH3T3 cells following infection, but none in the control (neo) vector.Foci were subjected to ultrathin section followed by Tuluidine bluestaining. FIG. 14A shows ultrathin sections of either control cells(FIG. 14A), while FIG. 14B shows Nsp1 transformed foci.

[0363] Three transformed foci of NIH3T3-MSCV.Nsp1 were picked andexpanded to generate sublines NIH3T3-MSCV.Nsp1-sc1, -sc2 and -sc3. 10⁷vector transfected control cells, untransformed NIH3T3.MSCV.Nsp1 andthree sublines were injected subcutaneously into the back of each nudemouse. Five mice were injected for each cell line. Tumor mass wasmeasured at two weeks and four weeks. The resultant tumors (four weekspost injection) were fixed, blocked and sections stained withhematoxylin and eosin. (FIG. 14C). No tumor growth was observed in anymouse injected with vector transfected control cells or untransformedNIH3T3-MSCV.Nsp1 cells. Every mouse injected with NIH3T3-MSCV.Nsp1-.sc1,-.sc2 or -.sc3 grew tumors. (FIG. 15).

Example 6 Apoptosis Resistance

[0364] Since the previous examples indicated that Nsp1 (SEQ ID NO:2)expression leads to NIH3T3 transformation and tumor formation,Applicants have investigated whether Nsp1 expression protects cells fromapoptosis induced by removal of growth factors. Subcloned cell lineswere derived from the morphologically transformed cellsNIH3T3-MSCV.Nsp1-.sub1 and -.sub2 (designated Nsp1.-sub1.1 andNsp1.sub2.1). These transformed clonal lines, the non-transformed cellculture (NIH3T3-Nsp1.non-trans.) and the control cells NIH3T3-neo wereserum starved for 48 hours in the presence or absence of the PI 3-kinaseinhibitor LY294002 and subjected to ANNEXIN V (Clontech) apoptosis assay(FIG. 16). Although the NIH3T3-Nsp1.non-trans. cells weremorphologically normal and did not form tumors in nude mice they weremore resistant to apoptosis induced by growth factor withdrawal thanwere the control NIH3T3-neo cells. This small but significant increasein resistance to apoptosis was abolished by the PI3 kinase inhibitorLY294002. In the vector control cells which do not express Nsp1 (SEQ IDNO:2), LY294002 did not by itself induce further apoptosis. In the Nsp1(SEQ ID NO:2) transformed sublines, there was an almost completeprotection from serum starvation induced apoptosis, but this effect wasnot sensitive to the treatment with the PI 3-kinase inhibitor. Thisdependent on PI 3-kinase at lower levels of Nsp1 (SEQ ID NO:1) wouldplace PI 3-kinase downstream of Nsp1 (SEQ ID NO:1). In contrast theobservation that the growth factor independence at high Nsp1 levels isnot inhibitable by LY294002 suggests that Nsp1 (SEQ ID NO:1) impacts anadditional pathway that functions independently of PI 3-kinase. That PI3-kinase is both necessary and sufficient for growth factor mediatedresistance to apoptosis had been previously reported. Kulik et al., Mol.Cell Biol. 17: 1595-606 (1997); Parrizas et al., J. Biol. Chem. 272:154-61 (1997); Vemuri et al., Development 122: 2529-37 (1996).

[0365] Material and Methods:

[0366] Control cells (NIH3T3-neo), non-transformed Nsp1 (SEQ ID NO:2)expressing cells (NIH3T3-Nsp1.non-trans.) and the transformed sublines(Nsp1.sub.1.1 and Nsp1.sub2.1) were serum starved in the presence ofabsence of 10 μg/ml LY294002 for 48 hours. The percent of apoptoticcells were assayed using ANNEXIN V-FITC (Clontech) on FACS according tothe manufacturers directions. Each cell line was assayed in triplicateand the means and standard deviations are shown. In transformedsublines, Nsp1 (SEQ ID NO:1) protected cells from serum starvationinduced apoptosis.

Example 7 PI 3-Kinase Interaction

[0367] In order to determine whether Nsp1 (SEQ ID NO:1) does interactwith PI 3-kinase, a GST fusion protein containing the PI 3-kinaseN-terminal or C-terminal SH2 domains were incubated with EGF treated oruntreated COS cell lysate transiently expressing Nsp1 (SEQ ID NO:2) orcontrols (FIG. 20). The C-terminal SH2 domain GST fusion protein doesinteract with Nsp1 (SEQ ID NO:1). This interaction appears to be atleast partially dependent on the phosphorylation status of Nsp1 (SEQ IDNO:1) as there is an increase in the amount of Nsp1 (SEQ ID NO:1) thatinteracts with PI3 kinase following EGF stimulation. The N-terminal SH2domain of PI3 kinase does not measureably interact with Nsp1 (SEQ IDNO:1).

[0368] Material and Methods:

[0369] COS cells transfected with pRK or with Nsp1 (SEQ ID NO:2) weretreated with 25 ng/ml EGF or left untreated. Cells were lysed in CoIPbuffer (supra) and incubated with PI 3-kinase N-terminal or C-terminalSH2 domain-GST beads (UBI). The precipitated Nsp1 (SEQ ID NO:1) wasdetected with anti-flag antibody.

Example 8 Use of PRO201, PRO308 or PRO309 as a Hybridization Probe

[0370] The following method describes use of a nucleotide sequenceencoding a PRO201, PRO308 or PRO309 polypeptide as a hybridizationprobe.

[0371] DNA comprising the coding sequence of a full-length or maturePRO201 (e.g., Nsp1), PRO308 (e.g., Nsp2) or PRO309 (e.g., Nsp3) and/orfragments thereof may be employed as a probe to screen for homologousDNAs (such as those encoding naturally-occurring variants of PRO201,PRO308 or PRO309 in human tissue cDNA libraries or human tissue genomiclibraries.

[0372] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled PRO201 (e.g., Nsp1), PRO308 (e.g., Nsp2)or PRO309 (e.g., Nsp3)-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% sodium pyrophosphate, 50 mMsodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

[0373] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO201, PRO308 or PRO309 can then beidentified using standard techniques known in the art.

Example 9 Expression of PRO201, PRO308 or PRO309 in E. coli

[0374] This example illustrates preparation of an unglycosylated form ofPRO201, PRO308 or PRO309 by recombinant expression in E. coli.

[0375] The DNA sequence encoding PRO201, PRO308 or PRO309 (SEQ ID NO:2,SEQ ID NO: 4, SEQ ID NO: 6) is initially amplified using selected PCRprimers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see Bolivar et al.,Gene, 2:95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a polyhis leader (includingthe first six STII codons, polyhis sequence, and enterokinase cleavagesite), the PRO201, PRO308 or PRO309 coding region, lambdatranscriptional terminator, and an argU gene.

[0376] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0377] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0378] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO201, PRO308 or PRO309 protein can then bepurified using a metal chelating column under conditions that allowtight binding of the protein.

[0379] Alternatively, expression in E coli may be performed usually thefollowing methology. The DNA sequence encoding PRO201 (e.g., Nsp1),PRO308 (e.g., Nsp2) or PRO309 (e.g., Nsp3) is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO201, PRO308 or PRO309 codingregion, lambda transcriptional terminator, and an argu gene.

[0380] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0381] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0382] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO201, PRO308 or PRO309 protein can then bepurified using a metal chelating column under conditions that allowtight binding of the protein.

Example 10 Expression of PRO201, PRO308 or PRO309 in Mammalian Cells

[0383] This example illustrates preparation of a glycosylated form ofPRO201, PRO308 or PRO309 by recombinant expression in mammalian cells.

[0384] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO201, PRO308 orPRO309 DNA is ligated into pRK5 with selected restriction enzymes toallow insertion of the PRO201, PRO308 or PRO309 DNA using ligationmethods such as described in Sambrook et al., supra. The resultingvector is called pRK5-PRO201, pRK5-PRO308 or pRK5-PRO309.

[0385] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO201, pRK5-PRO308 or pRK5-PRO309 DNA is mixed with about 1 μg DNAencoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] anddissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. Tothis mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and a precipitate is allowed to form for 10 minutesat 25° C. The precipitate is suspended and added to the 293 cells andallowed to settle for about four hours at 37° C. The culture medium isaspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds.The 293 cells are then washed with serum free medium, fresh medium isadded and the cells are incubated for about 5 days.

[0386] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/Ml ³⁵S-cysteine and 200 μCi/Ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO201, PRO308 or PRO309 polypeptide. Thecultures containing transfected cells may undergo further incubation (inserum free medium) and the medium is tested in selected bioassays.

[0387] In an alternative technique, PRO201, PRO308 or PRO309 may beintroduced into 293 cells transiently using the dextran sulfate methoddescribed by Somparyrac et al., Proc. Natl. Acad. Sci, 12:7575 (1981).293 cells are grown to maximal density in a spinner flask and 700 μgpRK5-PRO201, pRK5-PRO308 or pRK5-PRO309 DNA is added. The cells arefirst concentrated from the spinner flask by centrifugation and washedwith PBS. The DNA-dextran precipitate is incubated on the cell pelletfor four hours. The cells are treated with 20% glycerol for 90 seconds,washed with tissue culture medium, and re-introduced into the spinnerflask containing tissue culture medium, 5:g/ml bovine insulin and0.1:g/ml bovine transferrin. After about four days, the conditionedmedia is centrifuged and filtered to remove cells and debris. The samplecontaining expressed PRO201, PRO308 or PRO309 can then be concentratedand purified by any selected method, such as dialysis and/or columnchromatography.

[0388] In another embodiment, PRO201, PRO308 or PRO309 can be expressedin CHO cells. The pRK5-PRO201, PRO308 or PRO309 can be transfected intoCHO cells using known reagents such as CaPO₄ or DEAE-dextran. Asdescribed above, the cell cultures can be incubated, and the mediumreplaced with culture medium (alone) or medium containing a radiolabelsuch as ³⁵S-methionine. After determining the presence of PRO201, PRO308or PRO309 polypeptide, the culture medium may be replaced with serumfree medium. Preferably, the cultures are incubated for about 6 days,and then the conditioned medium is harvested. The medium containing theexpressed PRO201, PRO308 or PRO309 can then be concentrated and purifiedby any selected method.

[0389] Epitope-tagged PRO201, PRO308 or PRO309 may also be expressed inhost CHO cells. The PRO201, PRO308 or PRO309 may be subcloned out of thepRK5 vector. The subclone insert can undergo PCR to fuse in frame with aselected epitope tag such as a poly-his tag into a Baculovirusexpression vector. The poly-his tagged PRO201, PRO308 or PRO309 insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CHOcells can be transfected (as described above) with the SV40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO201, PRO308 or PRO309 can then be concentrated and purified by anyselected method, such as by Ni²⁺-chelate affinity chromatography.

Example 11 Expression of PRO201, PRO308 or PRO309 in Yeast

[0390] The following method describes recombinant expression of PRO201,PRO308 or PRO309 in yeast.

[0391] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO201, PRO308 or PRO309 from the ADH2/GAPDHpromoter. DNA encoding PRO201, PRO308 or PRO309, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression ofPRO201, PRO308 or PRO309. For secretion, DNA encoding PRO201, PRO308 orPRO309 can be cloned into the selected plasmid, together with DNAencoding the ADH2/GAPDH promoter, the yeast alpha-factor secretorysignal/leader sequence, and linker sequences (if needed) for expressionof PRO201, PRO308 or PRO309.

[0392] Yeast cells, such as yeast strain AB 110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0393] Recombinant PRO201, PRO308 or PRO309 can subsequently be isolatedand purified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing PRO201, PRO308 or PRO309may further be purified using selected column chromatography resins.

Example 12 Expression of PRO201, PRO308 or PRO309 inBaculovirus-Infected Insect Cells

[0394] The following method describes recombinant expression of PRO201,PRO308 or PRO309 in Baculovirus-infected insect cells.

[0395] The PRO201, PRO308 or PRO309 is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO201, PRO308 or PRO309 or the desired portion of the PRO201, PRO308 orPRO309 (such as the sequence encoding the extracellular domain of atransmembrane protein) is amplified by PCR with primers complementary tothe 5′ and 3′ regions. The 5′ primer may incorporate flanking (selected)restriction enzyme sites. The product is then digested with thoseselected restriction enzymes and subcloned into the expression vector.

[0396] Recombinant baculovirus can be generated by co-transfecting theabove plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression may be performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0397] Following PCR amplification, the respective coding sequences aresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand Baculogold® baculovirus DNA (Pharmingen) were co-transfected into105 Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711), usingLipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are modifications of thecommercially available baculovirus expression vector pVL1393(Pharmingen), with modified polylinker regions to include the His or Fctag sequences. The cells are grown in Hink's TNM-FH medium supplementedwith 10% FBS (Hyclone). Cells are incubated for 5 days at 28° C. Thesupernatant is harvested and subsequently used for the first viralamplification by infecting Sf9 cells in Hink's TNM-FH mediumsupplemented with 10% FBS at an approximate multiplicity of infection(MOI) of 10. Cells are incubated for 3 days at 28° C. The supernatant isharvested and the expression of the constructs in the baculovirusexpression vector determined by batch binding of 1 ml of supernatant to25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

[0398] The first viral amplification supernatant can be used to infect aspinner culture (500 ml) of Sf9 cells grown in ESF-921 medium(Expression Systems LLC) at an approximate MOI of 0.1. Cells wereincubated for 3 days at 28° C. The supernatant was harvested andfiltered. Batch binding and SDS-PAGE analysis is repeated, as necessary,until expression of the spinner culture is confirmed.

[0399] Expressed poly-his tagged PRO201, PRO308 or PRO309 can then bepurified, for example, by Ni²⁺-chelate affinity chromatography asfollows. Extracts are prepared from recombinant virus-infected Sf9 cellsas described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9;12.5 mM MgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), andsonicated twice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 Fm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO201, PRO308 or PRO309 are pooledand dialyzed against loading buffer.

[0400] Alternatively, purification of the IgG tagged (or Fc tagged)PRO201, PRO308 or PRO309 can be performed using known chromatographytechniques, including for instance, Protein A or protein G columnchromatography.

[0401] Alternatively, a modified baculovirus procedure may be usedincorporating high 5 cells. In this procedure, the DNA encoding thedesired sequence was amplified with suitable systems, such as Pfu(Stratagene), or fused upstream (5′-of) of an epitope tag contained witha baculovirus expression vector. Such epitope tags include poly-his tagsand immunoglobulin tags (like Fe regions of IgG). A variety of plasmidsmay be employed, including plasmids derived from commercially availableplasmids such as pIE1-1 (Novagen). The pIE1-1 and pIE1-2 vectors aredesigned for constitutive expression of recombinant proteins from thebaculovirus ie1 promoter in stably-transformed insect cells (1). Theplasmids differ only in the orientation of the multiple cloning sitesand contain all promoter sequences known to be important forie1-mediated gene expression in uninfected insect cells as well as thehr5 enhancer element. pIE1-1 and pIE1-2 include the ie translationinitiation site and can be used to produce fusion proteins. Briefly, thedesired sequence or the desired portion of the sequence (such as thesequence encoding the extracellular domain of a transmembrane protein)is amplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product was then digested with those selected restrictionenzymes and subcloned into the expression vector. For example,derivatives of pIE1-1 can include the Fc region of human IgG (pb.PH.IgG)or an 8 histidine (pb.PH.His) tag downstream (3′-of) the desiredsequence. Preferably, the vector construct is sequenced forconfirmation.

[0402] Hi5 cells are grown to a confluency of 50% under the conditionsof, 27° C., no CO₂, NO pen/strep. For each 150 mm plate, 30 ug of pIEbased vector containing the sequence was mixed with 1 ml Ex-Cell medium(Media: Ex-Cell 401+1/100 L-Glu JRH Biosciences #14401-78P (note: thismedia is light sensitive)), and in a separate tube. 100 ul of CellFectin(CelIFECTIN (GibcoBRL #10362-010) (vortexed to mix)) was mixed with 1 mlof Ex-Cell medium. The two solutions were combined and allowed toincubate at room temperature for 15 minutes. 8 ml of Ex-Cell media wasadded to the 2 ml of DNA/CellFECTIN mix and this is layered on Hi5 cellsthat have been washed once with Ex-Cell media. The plate is thenincubated in darkness for 1 hour at room temperature. The DNA/CellFECTINmix is then aspirated, and the cells are washed once with Ex-Cell toremove excess CellFECTIN 0.30 ml of fresh Ex-Cell media was added andthe cells are incubated for 3 days at 28C. The supernatant was harvestedand the expression of the sequence in the baculovirus expression vectorwas determined by batch binding of 1 ml of supernatent to 25 mL ofNi-NTA beads (QIAGEN) for histidine tagged proteins or Protein-ASepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed bySDS-PAGE analysis comparing to a known concentration of protein standardby Coomassie blue staining.

[0403] The conditioned media from the transfected cells (0.5 to 3 L) washarvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteincomprising the sequence is purified using a Ni-NTA column (Qiagen).Before purification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media was pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 48C. Afterloading, the column was washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein was then subsequently desaltedinto a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4%mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

[0404] Immunoadhesin (Fc containing) constructs of proteins are purifiedfrom the conditioned media as follows. The conditioned media is pumpedonto a 5 ml Protein A column (Pharmacia) which has been previouslyequilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, thecolumn is washed extensively with equilibration buffer before elutionwith 100 mM citric acid, pH 3.5. The eluted protein is immediatelyneutralized by collecting 1 ml fractions into tubes containing 275 mL of1 M Tris buffer, pH 9. The highly purified protein is subsequentlydesalted into storage buffer as described above for the poly-His taggedproteins. The homogeneity of the sequence is assessed by SDSpolyacrylamide gels and by N-terminal amino acid sequencing by Edmandegradation and other analytical procedures as desired or necessary.

Example 13 Preparation of Antibodies that Bind PRO201, PRO308 or PRO309

[0405] This example illustrates preparation of monoclonal antibodiesthat can specifically bind PRO201, PRO308 or PRO309.

[0406] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO201, PRO308 or PRO309, fusionproteins containing PRO201, PRO308 or PRO309, and cells expressingrecombinant PRO201, PRO308 or PRO309 on the cell surface. Selection ofthe immunogen can be made by the skilled artisan without undueexperimentation.

[0407] Mice, such as Balb/c, are immunized with the PRO201, PRO308 orPRO309 immunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing in ELISAassays to detect PRO201, PRO308 or PRO309 antibodies.

[0408] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO201, PRO308 or PRO309. Three to four days later, themice are sacrificed and the spleen cells are harvested. The spleen cellsare then fused (using 35% polyethylene glycol) to a selected murinemyeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597.The fusions generate hybridoma cells which can then be plated in 96 welltissue culture plates containing HAT (hypoxanthine, aminopterin, andthymidine) medium to inhibit proliferation of non-fused cells, myelomahybrids, and spleen cell hybrids.

[0409] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO201, PRO308 or PRO309. Determination of “positive” hybridomacells secreting the desired monoclonal antibodies against PRO201, PRO308or PRO309 is within the skill in the art.

[0410] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing theanti-PRO201, anti-PRO308 or anti-PRO309 monoclonal antibodies.Alternatively, the hybridoma cells can be grown in tissue culture flasksor roller bottles. Purification of the monoclonal antibodies produced inthe ascites can be accomplished using ammonium sulfate precipitation,followed by gel exclusion chromatography. Alternatively, affinitychromatography based upon binding of antibody to protein A or protein Gcan be employed.

Example 14 Gene Amplification

[0411] This example shows that the PRO201, PRO308 or PRO309-encodinggenes are amplified in the genome of certain human lung, colon and/orbreast cancers and/or cell lines. Amplification is associated withoverexpression of the gene product, indicating that the bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for the PRO201, PRO308 or PRO309proteins are useful targets for therapeutic intervention in certaincancers such as colon, lung, breast and other cancers. Therapeutic agentmay take the form of antagonists of binding specificities for at leasttwo different antigens. In the present case, one of the bindingspecificities is for the PRO201-, PRO308- or PRO309-encoding genes, forexample, murine-human chimeric, humanized or human antibodies against abinding specificities for at least two different antigens. In thepresent case, one of the binding specificities is for the PRO201, PRO308or PRO309 polypeptide.

[0412] The starting material for the screen was genomic DNA isolatedfrom a variety cancers. The DNA is quantitated precisely, e.g.fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm 7700 Sequence Detection System™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding binding specificities for at leasttwo different antigens. In the present case, one of the bindingspecificities is for the PRO201, PRO308 or PRO309 is over-represented inany of the primary lung or colon cancers or cancer cell lines or breastcancer cell lines that were screened. The primary lung cancers wereobtained from individuals with tumors of the type and stage as indicatedin Table 1. An explanation of the abbreviations used for the designationof the primary tumors listed in Table 1 and the primary tumors and celllines referred to throughout this example has been given hereinbefore.

[0413] The results of the Taqman™ are reported in delta (A) CT units.One unit corresponds 1 PCR cycle or approximately a 2-fold amplificationrelative to normal, two units corresponds to 4-fold, 3 units to 8-foldamplification and so on. Quantitation was obtained using primers and aTaqman™ fluorescent prove derived from the binding specificities for atleast two different antigens. In the present case, one of the bindingspecificities is for the PRO201-, PRO308- and PRO309-encoding gene.Regions of binding specificities for at least two different antigens. Inthe present case, one of the binding specificities is for the PRO201,PRO308 or PRO309 which are most likely to contain unique nucleic acidsequences and which are least likely to have spliced out introns arepreferred for the primer and probe derivation, e.g. 3-untranslatedregion. The sequences for the primers and probes (forward, reverse andprobe) used for the PRO201, PRO308 or PRO309 gene amplification were asfollows: PRO201 (DNA30676): 30676.tm.f 5′-CGCAGACACCCTTCTTCACA-3′ (SEQID NO:24) 30676.tm.r 5′-CGACTCCTTTGGTCTCTTCTGG-3′ (SEQ ID NO:25)30676.tm.p 5′-CCGGGACCCCCAGGTTTTTGC-3′ (SEQ ID NO:26) DNA40556:40556.tm.f: 5′-AGGGTCCTGCGTGGACTCT-3′ (SEQ ID NO:27) 40556.tm.r:5′-TCCTGTTCTTCCTCAATGGAGAC-3′ (SEQ ID NO:28) 40556.tm.p: 5′-CCATCCCACCTGCTACATGCTCACC-3′ (SEQ ID NO:29)

[0414] The 5′ nuclease assay reaction is a fluorescent PCR-basedtechnique which makes use of the 5′ exonuclease activity of Taq DNApolymerase enzyme to monitor amplification in real time. Twooligonucleotide primers are used to generate an amplicon typical of aPCR reaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the TAQ DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

[0415] The 5′ nuclease procedure is run on a real-time quantitative PCRdevice such as the ABI Prism 7700TM Sequence Detection. The systemconsists of a thermocycler, laser, charge-coupled device (CCD) cameraand computer. The system amplifies samples in a 96-well format on athermocycler. During amplification, laser-induced fluorescent signal iscollected in real-time through fiber optics cables for all 96 wells, anddetected at the CCD. The system includes software for running theinstrument and for analyzing the data.

[0416] 5′ Nuclease assay data are initially expressed as Ct, or thethreshold cycle. This is defined as the cycle at which the reportersignal accumulates above the background level of fluorescence. The ΔCtvalues are used as quantitative measurement of the relative number ofstarting copies of a particular target sequence in a nucleic acid samplewhen comparing cancer DNA results to normal human DNA results.

[0417] Table 1 describes the stage, T stage and N stage of variousprimary tumors which were used to screen the PRO201, PRO308 or PRO309compounds of the invention. TABLE 1 Primary Lung and Colon TumorProfiles Other Dukes T N Primary Tumor Stage Stage Stage Stage StageHuman lung tumor SqCCA IB — — T1 N1 (SRCC724) [LT1] Human lung tumorNSCCa IA — — T3 N0 (SRCC725) [LT1a] Human lung tumor AdenoCa IB — — T2N0 (SRCC726) [LT2] Human lung tumor AdenoCa IB — — T1 N2 (SRCC727) [LT3]Human lung tumor SqCCq IIB — — T2 N0 (SRCC728) [LT4] Human lung tumorAdenoCa IV — — T1 N0 (SRCC729) [LT6] Human lung tumor Aden/SqCCa IB — —T1 N0 (SRCC730) [LT7] Human lung tumor AdenoCa IIB — — T2 N0 (SRCC731)[LT9] Human lung tumor SqCCa IA — — T2 N1 (SRCC732) [LT10] Human lungtumor AdenoCa IB — — T1 N1 (SRCC733) [LT11] Human lung tumor AdenoCa IIA— — T2 N0 (SRCC734) [LT12] Human lung tumor BAC IB — — T2 N0 (SRCC735)[LT13] Human lung tumor SqCCa IB — — T2 N0 (SRCC736) [LT15] Human lungtumor SqCCa IIB — — T2 N0 (SRCC737) [LT16] Human lung tumor SqCCa IIB —— T2 N1 (SRCC738) [LT17] Human lung tumor SqCCa IB — — T2 N0 (SRCC739)[LT18] Human lung tumor SqCCa IB — — T2 N0 (SRCC740) [LT19] Human lungtumor LCCa IIB — — T3 N1 (SRCC741) [LT21] Human colon AdenoCa — M1 D pT4N0 (SRCC742) [CT2] Human colon AdenoCa — B pT3 N0 (SRCC743) [CT3] Humancolon AdenoCa B T3 N0 (SRCC744) [CT8] Human colon AdenoCa A pT2 N0(SRCC745) [CT10] Human colon AdenoCa MO, R1 B T3 N0 (SRCC746) [CT12]Human colon AdenoCa PMO, B pT3 pN0 (SRCC747) [CT14] RO Human colonAdenoCa M1, R2 D T4 N2 (SRCC748) [CT15] Human colon AdenoCa PMO B pT3pN0 (SRCC749) [CT16] Human colon AdenoCa C1 pT3 pN1 (SRCC750) [CT17]Human colon AdenoCa MO, R1 B pT3 N0 (SRCC751) [CT1] Human colon AdenoCaB pT3 M0 (SRCC752) [CT4] Human colon AdenoCa G2 C1 pT3 pN0 (SRCC753)[CT5] Human colon AdenoCa PMO, B pT3 pN0 (SRCC754) [CT6] RO Human colonAdenoCa G1 A pT2 pN0 (SRCC755) [CT7] Human colon AdenoCa G3 D pT4 pN2(SRCC756) [CT9] Human colon AdenoCa B T3 N0 (SRCC757) [CT11] Human colonAdenoCa MO, B pT3 pN0 (SRCC758) [CT18] RO

[0418] DNA Preparation:

[0419] DNA was prepared from cultured cell lines, primary tumors, normalhuman blood. The isolation was performed using purification kit, bufferset and protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

[0420] Cell Culture Lysis:

[0421] Cells were washed and trypsinized at a concentration of 7.5×10⁸per tip and pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2×with PBS. The cells were then suspended into 10 mL PBS. BufferCl was equilibrated at 4° C. Quiagen protease #19155 was diluted into6.25 ml cold ddH₂O to a final concentration of 20 mg/ml and equilibratedat 4° C. 10 mL of G2 Buffer was prepared by diluting Quiagen RNAse Astock (100 mg/ml) to a final concentration of 200 μg/ml.

[0422] Buffer C1 (10 mL, 4° C.) and ddH2O (40 mL, 4° C.) were then addedto the 10 mL of cell suspension, mixed by inverting and incubated on icefor 10 minutes. The cell nuclei were pelleted by centrifuging in aBeckman swinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 mL Buffer Cl (at 4° C.) and 6 mL ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30-60 minutes, pelleting at 3000×g for 10 min., 4°C.).

[0423] Solid Human Tumor Sample Preparation and Lysis:

[0424] Tumor samples were weighed and placed into 50 ml conical tubesand held on ice. Processing was limited to no more than 250 mg tissueper preparation (I tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood to order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

[0425] Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

[0426] Human Blood Preparation and Lysis:

[0427] Blood was drawn from healthy volunteers using standard infectiousagent protocols and citrated into 10 ml samples per tip. Quiagenprotease was freshly prepared by dilution into 6.25 ml cold ddH₂O to afinal concentration of 20 mg/ml and stored at 4° C. G2 buffer wasprepared by diluting RNAse A to a final concentration of 200 μg/ml from100 mg/ml stock. The blood (10 ml) was placed into a 50 ml conical tubeand 10 ml Cl buffer and 30 ml ddH₂O (both previously equilibrated to 4°C.) were added, and the components mixed by inverting and held on icefor 10 minutes. The nuclei were pelleted with a Beckman swinging bucketrotor at 2500 rpm, 4° C. for 15 minutes and the supernatant discarded.With a vortex, the nuclei were suspended into 2 ml Cl buffer (4° C.) and6 ml ddH₂O (4° C.). Vortexing was repeated until the pellet was white.The nuclei were then suspended into the residual buffer using a 200 μltip. G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

[0428] Purification of Cleared Lysates:

[0429] (1) Isolation of Genomic DNA:

[0430] Genomic DNA was equilibrated (1 sample per maxi tip preparation)with 10 ml QBT buffer. QF elution buffer was equilibrated at 50° C. Thesamples were vortexed for 30 seconds, then loaded onto equilibrated tipsand drained by gravity. The tips were washed with 2×15 ml QC buffer. TheDNA was eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with15 ml QF buffer (50° C.). Isopropanol (10.5 ml) was added to eachsample, the tubes covered with parafin and mixed by repeated inversionuntil the DNA precipitated. Samples were pelleted by centrifugation inthe SS-34 rotor at 15,000 rpm for 10 minutes at 4° C. The pelletlocation was marked, the supernatant discarded, and 10 ml 70% ethanol(4° C.) was added. Samples were pelleted again by centrifugation on theSS-34 rotor at 10,000 rpm for 10 minutes at 4° C. The pellet locationwas marked and the supernatant discarded. The tubes were then placed ontheir side in a drying rack and dried 10 minutes at 37° C., taking carenot to overdry the samples.

[0431] After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5)and placed at 50° C. for 1-2 hours. Samples were held overnight at 4° C.as dissolution continued. The DNA solution was then transferred to 1.5ml tubes with a 26 gauge needle on a tuberculin syringe. The transferwas repeated 5×in order to shear the DNA. Samples were then placed at50° C. for 1-2 hours.

[0432] (2) Quantitation of Genomic DNA and Preparation for GeneAmplification Assay:

[0433] The DNA levels in each tube were quantified by standard A260,A280 spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) usingthe 0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer.A260/A280 ratios were in the range of 1.8-1.9. Each DNA samples was thendiluted further to approximately 200 ng/ml in TE (pH 8.5). If theoriginal material was highly concentrated (about 700 ng/μl), thematerial was placed at 50° C. for several hours until resuspended.

[0434] Fluorometric DNA quantitation was then performed on the dilutedmaterial (20-600 ng/ml) using the manufacturer's guidelines as modifiedbelow. This was accomplished by allowing a Hoeffer DyNA Quant 200fluorometer to warm-up for about 15 minutes. The Hoechst dye workingsolution (#H33258, 10 μl, prepared within 12 hours of use) was dilutedinto 100 ml 1×TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

[0435] The fluorometricly determined concentration was then used todilute each sample to 10 ng/μl in ddH₂O. This was done simultaneously onall template samples for a single TaqMan plate assay, and with enoughmaterial to run 500-1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 CT. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8-9 plates or 64 tests.

[0436] Gene Amplification Assay:

[0437] The PRO201, PRO308 or PRO309 compounds of the invention werescreened in the following primary tumors and the resulting ΔCt valuesare reported in Table 2. TABLE 2 ΔCt values of DNA30676 (SEQ ID NO:2)and DNA40556 (SEQ ID NO:20) in selected primary lung and colon tumorsand cell lines Primary Tumor or Cell Line DNA30676 DNA40556 LT1 0.27,0.05, 0.41, −0.27 0.31, −0.14, 0.54 LT1a 1.15, 0.99, 1.37, 0.9 1.44,0.6, 1.06 LT2 0.22, 0.24, −0.1, 0.04 0.65, 0.33 LT3 2.16, 1.89, 2.52,1.63 1.21, 0.35, 0.26 LT4 0.22, 0.03, 0.41, 0.16 1.38, 0.79 LT6 1.66,0.76, 0.82, −0.02 0.75, −0.02, 0.27 LT7 1.01, 0.54, 0.69, −0.33 1.03,−0.08, 0.88 LT8 — 1.14 LT9 1.16, 0.74, 1.11, 0.39 1.32, 0.16, 0.40 LT101.78, 0.72, 1.22, 0.6 1.0, 0.12, 0.40 LT11 1.86, 1.42, 1.29 1.985, 0.67LT12 0.81, 0.69, 0.21 1.725, −0.49, 0.27 LT13 2.71, 2.24, 1.45 1.485,0.51, 1.035 LT15 2.89, 2.79, 2.07 1.965, 0.58, 0.975 LT16 1.13, 0.4,1.78 0.775, 0.33, 0.595 LT17 2.11, 1.55, 1.23 1.965, 0.04 LT18 0.22,−0.08, 1.72 0.345, −0.07 LT19 3.51, 3.23 1.425 LT21 1.9, 0.7 1.085 LT22−0.13 −2.65 CT2 3.81 0.385, 0.865 CT3 2.3 0.545, 0.315 CT8 1.97 0.695,0.305 CT10 3.01 1.135, 0.845 CT12 2.76 0.925, 0.675 CT14 3.34 1.245,0.995 CT15 2.58 0.895 CT16 2.46 0.755, 0.635 CT17 2.64 0.565, 0.425 CT11.57 0.875, 0.865 CT4 3.39 0.585, 0.585 CT5 2.95 1.005 CT6 2.81 0.995CT7 2.77 0.795 CT9 2.63 0.575, 0.805 CT11 3 1.135 CT18 2.23 0.755 A549 —1.255 Calu-1 2.37 — Calu-6 2.61 — H157 2.39 — H441 2.07 — H460 0.28 —SKMES1 3.01 — H522 2.91 — H810 2.07 — SW620 0.69 — Colo320 1.95 0.685HT29 0.37 — SKCO1 0.3 — SW403 0.59 — LS174T 0.38 — Colo-205 1.26 — HCT150.61 — HCT116 — 0.935 HCC2998 0.27 — KM12 0.82 —

[0438] DNA30676 (SEQ ID NO:2) was also reexamined along with selectedtumors from the above initial screen with framework mapping. FIG. 18 andTable 3 indicate the chromosomal mapping of the framework markers thatwere used in the present example. The framework markers are locatedapproximately every 20 megabases and were used to control aneuploidy.

[0439] DNA30676 (SEQ ID NO:2) was also reexamined with epicentermapping. The markers indicated in Tables 4 are located in closeproximity (in the genome) to DNA30676 (SEQ ID NO:2) and are used toassess the relative amplification in the immediate vicinity ofChromosome 19 wherein the respective molecule is located. The distancebetween individual markers is measured in centirays (cR), which is aradiation breakage unit approximately equal to a 1% chance of a breakagebetween two markers. One cR is very roughly equivalent to 20 kilobases.The marker SHGC-35441 is the marker found to be the closest to thelocation on chromosome 19 where DNA30676 maps. TABLE 3 Framework MarkersAlong Chromosome 19 Map Position on Chromosome 19 Stanford Human GenomeCenter Marker Name S12 AFMa107xc9 S50 SHGC-31335 S105 SHGC-34102 S155SHGC-16175

[0440] TABLE 4 Epicenter Markers Along Chromosome 19 used for DNA30676Map Position on Stanford Human Genome Distance to next Chromosome 19Center Marker Name Marker (cR) S12 AFMa107xc9 22 S16 SHGC-1261 53 S17SHGC-2897 7 S18 SHGC-35441 59 S19 SHGC-6150 33 S21 AFM224ye9 21 S23SHGC-31478 25 S24 SHGC-3921 —

[0441] The ΔCt values of the above described framework markers alongChromosome 19 relative to PRO201 is indicated for selected tumors inTable 5. TABLE 5 Amplification of framework markers relative to DNA30676(ΔCt) Framework Markers Tumor S12 DNA30676 S50 S105 S155 LT1 0.16 −0.180.06 −0.42 0.11 LT1a 0.05 0.79 −0.27 0.17 0.40 LT2 0.48 −0.09 0.41 0.520.13 LT3 0.27 1.04 0.83 0.11 0.50 LT4 0.48 −0.18 0.67 0.20 0.56 LT6 0.72−0.23 0.74 0.32 0.35 LT7 0.82 −0.36 0.85 0.95 0.95 LT9 0.72 −0.75 0.610.19 0.64 LT10 0.82 0.05 0.98 0.62 0.53 LT11 0.13 0.64 0.25 0.55 −0.34LT12 0.04 −0.60 0.60 0.21 −0.17 LT13 −0.06 0.67 0.57 −0.30 −0.05 LT15−0.03 1.43 −0.77 0.12 −0.04 LT16 0.46 1.35 1.37 0.51 0.23 LT17 0.37 1.510.74 0.21 0.22 LT18 0.39 1.22 0.57 0.11 0.16 LT22 0.79 0.13 0.76 −0.050.16 CT2 0.25 2.81 0.29 0.37 −0.02 CT3 −0.17 2.03 −0.10 0.34 −0.28 CT80.13 1.39 0.57 0.18 −0.16 CT10 0.15 2.21 0.51 −0.01 −0.81 CT12 0.13 1.930.57 0.41 0.20 CT14 0.40 2.37 0.39 0.45 0.36 CT15 −0.23 1.27 −0.30 −0.060.56 CT16 0.38 1.76 0.31 0.24 0.04 CT17 0.25 1.65 0.71 0.32 0.22

[0442] Table 6 indicate the ΔCt values for results of epicenter mappingrelative to DNA30676 (SEQ ID NO:2), indicating the relativeamplification in the region more immediate to the actual location ofDNA30676 (SEQ ID NO:2) along chromosome 19. TABLE 6 Amplification ofepicenter markers relative to DNA30676 (ΔCt) Tumor S12 S16 S17 S18DNA30676 S21 S23 S24 LT1 — 0.22 −0.16 0.02 −0.29 0.40 −0.02 0.14 LT1a —0.11 −0.52 0.32 0.58 −0.55 0.04 −0.15 LT2 — −0.07 −0.07 0.34 −0.04 0.070.13 0.12 LT3 — 0.01 −0.46 0.47 1.87 0.16 0.24 0.02 LT4 — −0.36 −0.960.93 −1.18 −0.54 −0.07 −0.23 LT6 — −0.35 −0.70 −0.04 0.28 −0.24 −0.12−0.01 LT7 — −0.32 −0.34 −0.27 0.29 −0.74 −0.07 0.05 LT9 — −0.42 −0.66−0.36 0.07 −1.42 −0.26 −0.70 LT10 — −0.26 −0.14 −0.07 0.55 −0.32 −0.04−0.08 LT11 — −0.22 −0.77 0.05 0.68 −0.85 −0.13 0.09 LT12 — −0.94 −1.52−1.26 0.13 0.08 −0.09 0.24 LT13 — 0.24 0.02 0.35 1.44 −0.08 0.50 0.49LT15 — −0.09 −0.64 0.26 1.99 0.03 0.09 −0.06 LT16 — 0.06 −0.16 0.20 1.720.75 0.54 0.64 LT17 — −0.91 −1.71 −0.78 −0.15 −2.89 −0.82 −0.42 LT18 —0.30 −0.20 0.71 1.09 −0.29 0.34 0.80 LT22 — 0.37 −0.82 0.47 0.07 0.460.38 0.65 CT1 0.18 0.02 0.32 0.57 1.61 0.75 0.56 0.05 CT2 0.46 0.19 0.350.59 3.51 −0.15 0.53 0.14 CT3 −0.02 −0.24 0.05 0.13 2.19 −0.31 0.13−0.34 CT4 0.29 0.20 0.42 0.64 3.22 0.47 0.27 0.33 CT5 −0.15 −0.16 0.12−0.21 2.83 0.09 −0.08 −0.17 CT6 0.13 0.17 0.87 0.26 2.93 0.44 0.04 0.39CT7 0.13 −0.03 0.78 −0.04 2.43 −0.68 −0.26 0.20 CT8 0.45 −0.03 0.58 0.221.95 0.25 0.57 0.07 CT9 0.50 0.41 0.98 0.64 2.72 0.24 0.06 0.66 CT100.11 −0.40 0.32 0.13 3.12 −0.16 0.28 −0.16 CT11 0.18 0.01 0.45 0.82 3.260.34 0.00 0.27 CT12 0.53 0.08 0.72 0.40 2.77 0.36 0.67 0.09 CT14 0.57−0.13 0.87 0.63 2.88 0.59 0.74 0.09 CT15 −0.09 −0.57 0.05 0.11 2.60−0.07 0.20 −0.34 CT16 0.57 −0.21 0.80 0.36 2.61 0.38 0.49 0.16 CT17 0.25−0.26 0.38 0.29 2.24 −0.05 0.67 0.05 CT18 0.38 0.18 0.53 0.49 2.48 0.41−0.29 0.12

DISCUSSION and CONCLUSION

[0443] The ΔCt values for DNA30676 (SEQ ID NO:2) and DNA40556 (SEQ IDNO:20) in a variety of lung and colon tumors are reported in Table 2. AΔCt of >1 was typically used as the threshold value for amplificationscoring, as this represents a doubling of gene copy. Table 2 indicatesthat significant amplification of DNA30676 occurred in: primary lungtumors: LT1a, LT3, LT11, LT13, LT15, LT17, LT19; primary colon tumors,CT2, CT3, CT8, CT10, CT12, CT14, CT15, CT16, CT17, CT1, CT4, CT5, CT6,CT7, CT9, CT11, CT18; lung tumor cell lines Calu-1, Calu-6, H1157, H441,SKMES1, H522 and H1810; and colon tumor cell lines Colo-320 andColo-205. Table 2 further indicates significant amplification ofDNA40556 in: primary lung tumors LTI a, LT8, LT11, LT15, LT17, LT19;primary colon tumors CT14, CT5, CT11 and lung tumor cell line A549.

[0444] The ΔCt and average ΔCt values of DNA30676 (SEQ ID NO:2) for theprimary lung tumor hits are: 1.10, 2.05, 1.52, 2.13, 2.58, 1.63, 3.42;primary colon tumor hits: 3.81, 2.3, 1.97, 3.01, 2.76, 3.34, 2.58, 2.46,2.64, 1.57, 3.39, 2.95, 2.81, 2.77, 2.63, 3.0, 2.23; lung tumor cellline hits: 2.37, 2.61, 2.39, 2.07, 3.01, 2.91 and 2.07; colon tumor celllines: 1.95 and 1.26. The ΔCt and average ΔCt values of DNA40556 for theprimary lung hits are 1.03, 1.14, 1.33, 1.18, 1.00 and 1.42; colontumorhits: 1.12, 1.00 and 1.135; lung tumorcell line 1.26.

[0445] The ΔCt and average ΔCt values of DNA30676 (SEQ ID NO:2)enumerated in the previous paragraph represent an increase in gene copy,relative to normal tissue, of 2.14, 4.14, 2.87, 4.38, 5.98, 3.09, 10.70for the primary lung tumor hits: 14.03, 4.92, 3.92, 8.06, 6.77, 10.13,5.98, 5.50, 6.23, 2.97, 10.48, 7.73, 7.01, 6.82, 6.19, 8.0, 4.69 for theprimary colon tumor hits; 5.17, 6.10, 5.24, 4.20, 8.06, 7.51, 4.20 forthe lung tumor cell line hits; 3.86, 2.40 for the colon tumor celllines. For DNA40556, these values represent an increase in gene copy,relative to normal tissue of: 2.04, 2.20, 2.51, 2.27, 2.0, 2.68 for thelung tumor hits, 2.17, 2.0, 2.20 for the colon tumor hits; 2.39.

[0446] Amplification has been confirmed by framework mapping forDNA30676 (SEQ ID NO:2): in primary lung tumors LT3, LT15, LT16, LT17,LT18; and in primary colon tumors CT2, CT3, CT8, CT9, CT10, CT12, CT14,CT15, CT16, CT17. The reported ΔCt values for the primary lung tumorsare 1.04, 1.43, 1.35, 1.51 and 1.22, while the primary colon tumorsreport 2.81, 2.03, 1.39, 2.21, 1.93, 2.37, 1.27, 1.76 and 1.65. Relativeto normal tissue, this represents approximately a 2.06, 2.69, 2.55,2.85, and 2.32 fold increase for the lung tumors, and a 7.01, 4.08,2.62, 4.63, 3.81, 5.17, 2.41, 3.39, 3.14 fold increase for the colontumors. Epicenter mapping for DNA30676 resulted in confirmation ofsignificant amplification: in primary lung tumors LT3, LT13, LT15, LT16,LT18; and in primary colon tumors CT1, CT2, CT3, CT4, CT5, CT6, CT7,CT8, CT9, CT10, CT11, CT12, CT14, CT15, CT16, CT17 and CT18. Thereported ΔCt values for the primary lung tumors were 1.87, 1.44, 1.99,1.72 and 1.09, while the primary colon tumors indicated ΔCt and averageΔCt values of 2.56, 3.51, 2.19, 3.22, 2.83, 2.93, 2.43, 1.95, 2.72,3.12, 3.26, 2.77, 2.88, 2.60, 2.61, 2.24 and 2.48. Relative to normaltissue, this represents a 3.66, 2.71, 3.97, 3.29, 2.13-fold increase ingene copy for the lung tumors and a 5.90, 11.39, 4.56, 9.32, 7.11, 7.62,5.39, 3.86, 6.59, 8.69, 9.58, 6.82, 7.36, 6.06, 6.11, 4.72, 5.58-foldincrease increase in gene copy for the colon tumors.

[0447] In contrast, the amplification of the closest known frameworkmarkers (with one exception, i e. S50)(Table 5) or epicenter markers(Table 6) does not occur to a greater extent than that of DNA30676 (SEQID NO:2). This strongly suggests that DNA30676 (SEQ ID NO:2) is the generesponsible for the amplification of the particular region on Chromosome19. Because amplification of DNA30676 (SEQ ID NO:2) occurs in variouslung and colon tumors and cell lines (especially colon), it is highlyprobable to play a significant role in tumor formation or growth. As aresult, antagonists (e.g., antibodies) directed against the proteinencoded by DNA30676 (SEQ ID NO:2)(i.e., Nsp1, SEQ ID NO:1), DNA40575(SEQ ID NO:4) and DNA61601 (SEQ ID NO:6) would be expected to haveutility in cancer therapy.

Example 15 In Situ Hybridization

[0448] In situ hybridization is a powerful and versatile technique forthe detection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

[0449] In situ hybridization was performed following an optimizedversion of the protocol by Lu and Gillett, Cell Vision 1: 169-176(1994), using PCR-generated ³³P-labeled riboprobes. Briefly,formalin-fixed, paraffin-embedded human tissues were sectioned,deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutesat 37° C., and further processed for in situ hybridization as describedby Lu and Gillett, supra. A [³³-P] UTP-labeled antisense riboprobe wasgenerated from a PCR product and hybridized at 55° C. overnight. Theslides were dipped in Kodak NTB2 nuclear track emulsion and exposed for4 weeks.

[0450]³³P-Riboprobe Synthesis

[0451] 6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol)were speed vac dried. To each tube containing dried ³³P-UTP, thefollowing ingredients were added:

[0452] 2.0 μl 5× transcription buffer

[0453] 1.0 μl DTT (100 mM)

[0454] 2.0 μl NTP mix (2.5 mM: 10 μl; each of 10 mM GTP, CTP & ATP+10 μlH₂O)

[0455] 1.0 μl UTP (50 μM)

[0456] 1.0 μl Rnasin

[0457] 1.0 μl DNA template (1 μg)

[0458] 1.0 μl H₂O

[0459] 1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

[0460] The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNasewere added, followed by incubation at 37° C. for 15 minutes. 90 μl TE(10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture waspipetted onto DE81 paper. The remaining solution was loaded in aMicrocon-50 ultrafiltration unit, and spun using program 10 (6 minutes).The filtration unit was inverted over a second tube and spun usingprogram 2 (3 minutes). After the final recovery spin, 100 μl TE wereadded. 1 μl of the final product was pipetted on DE81 paper and countedin 6 ml of Biofluor II.

[0461] The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μlof RNA Mrk III were added to 3 μμl of loading buffer. After heating on a95° C. heat block for three minutes, the gel was immediately placed onice. The wells of gel were flushed, the sample loaded, and run at180-250 volts for 45 minutes. The gel was wrapped in saran wrap andexposed to XAR film with an intensifying screen in −70° C. freezer onehour to overnight.

[0462]³³P-Hybridization

[0463] Pretreatment of frozen sections The slides were removed from thefreezer, placed on aluminium trays and thawed at room temperature for 5minutes. The trays were placed in 55° C. incubator for five minutes toreduce condensation. The slides were fixed for 10 minutes in 4%paraformaldehyde on ice in the fume hood, and washed in 0.5×SSC for 5minutes, at room temperature (25 ml 20×SSC+975 ml SQ H₂O). Afterdeproteination in 0.5 μg/ml proteinase K for 10 minutes at 37° C. (12.5μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), thesections were washed in 0.5×SSC for 10 minutes at room temperature. Thesections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.

[0464] Pretreatment of paraffin-embedded sections The slides weredeparaffinized, placed in SQ H₂O, and rinsed twice in 2×SSC at roomtemperature, for 5 minutes each time. The sections were deproteinated in20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNasebuffer; 37° C., 15 minutes)—human embryo, or 8× proteinase K (100 μl in250 ml Rnase buffer, 37° C., 30 minutes)—formalin tissues. Subsequentrinsing in 0.5×SSC and dehydration were performed as described above.

[0465] Prehybridization The slides were laid out in plastic box linedwith Box buffer (4×SSC, 50% formamide)—saturated filter paper. Thetissue was covered with 50 μl of hybridization buffer (3.75 g DextranSulfate +6 ml SQ H₂O), vortexed and heated in the microwave for 2minutes with the cap loosened. After cooling on ice, 18.75 ml formamide,3.75 ml 20×SSC and 9 ml SQ H₂O were added, the tissue was vortexed well,and incubated at 42° C. for 1-4 hours.

[0466] Hybridization 1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock)per slide were heated at 95° C. for 3 minutes. The slides were cooled onice, and 48 μl hybridization buffer were added per slide. Aftervortexing, 50 μl ³³P mix were added to 50 μl prehybridization on slide.The slides were incubated overnight at 55° C.

[0467] Washes Washing was done 2×10 minutes with 2×SSC, EDTA at roomtemperature (400 ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4L), followed byRNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 mlRnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC,EDTA at room temperature. The stringency wash conditions were asfollows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA,V_(f)=4L).

[0468] Expression of DNA 30676 panc.shc (nsp-1) in Human Tissues III

[0469] Comparable background signal observed with sense and antisenseprobes in many tissues. The only sites where expression appeared to bespecific were fetal thymic medulla, fetal spleen, epithelium of fetalsmall intestine and in osteocytes at regions of new bone formation. Nospecific signal was observed in either fetal or normal adult pancreas.Fetal tissues were approximately 12-16 weeks gestation. Oligos used wereB-191D and B-191E.

[0470] Expression of DNA 30676 (SHC hlog/nsp1) in Colon Tumors, FetalLiver, and Transfected Cell Lines

[0471] The purpose of this study was to determine the expression of DNA30676 in colonic carcinomas. This DNA has been shown to be amplified incolon cancer. Expression was analyzed in control cell pellets and in 10colon cancers. Control cell pellets included SHC transfected 293 cellsand SW480 cells, which express SHC (cells were submitted as H98-717).

[0472] Examination of cell pellets showed the SHC transfected cells werepositive with both sense and antisense probes making interpretation ofthis study problematic. The SW480 cells were negative with both probes.For the colon cancers only AS probes were run. A number of the coloncancers showed slight expression, and this was strongest in specimen9727/98. However, in light of the cell pellet data this signal isdifficult to interpret and overall, it is felt that the signal isinsufficient to be called as positive.

[0473] The oligonucleotide probes indicated below were used in the insitu hybridizations described in this example:B166A-:GGATTCTAATACGACTCACTATAGGGCGCGGAGGCTGCTCTGGGGTAG (SEQ ID NO:30)B166B-CTATGAAATTAACCCTCACTAAAGGGATGTTGCCCTGGCTGGTCTTGA (SEQ ID NO:31)B-191D-GGATTCTAATACGACTCACTATAGGGCATCTGCCTTGCCCCGAACGAG (SEQ ID NO:32)B-191E-CTATGAAATTAACCCTCACTAAAGGGATCATCCAGAGCCCGCATCAGC (SEQ ID NO:33)A-3231-GGATTCTAATACGACTCACTATAGGGCAGATGTGGAAGACTGAGGCCT (SEQ ID NO:34)A-323J-CTATGAAATTAACCCTCACTAAAGGGAATATGTGCCAAATCTGCAGGCT (SEQ ID NO:35)

[0474] Deposit of Material

[0475] The following materials have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC): Material ATCC Dep. No. Deposit Date DNA30676-1223 209567 Dec.23, 1997 DNA40575-1223 209565 Dec. 23, 1997 DNA40556-1223 209566 Dec.23, 1997 DNA40554-1223 209564 Dec. 23, 1997 DNA61601-1223 209713 Mar.31, 1998

[0476] DNA40556 & DNA40554 may be combined to cover a full length codingsequence of a Nsp3 variant.

[0477] This deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

[0478] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0479] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1 35 1 576 PRT Homo sapiens 1 Met Gln Val Pro Gln Asp Gly Glu Asp LeuAla Gly Gln Pro Trp 1 5 10 15 Tyr His Gly Leu Leu Ser Arg Gln Lys AlaGlu Ala Leu Leu Gln 20 25 30 Gln Asp Gly Asp Phe Leu Val Arg Ala Ser GlySer Arg Gly Gly 35 40 45 Asn Pro Val Ile Ser Cys Arg Trp Arg Gly Ser AlaLeu His Phe 50 55 60 Glu Val Phe Arg Val Ala Leu Arg Pro Arg Pro Gly ArgPro Thr 65 70 75 Ala Leu Phe Gln Leu Glu Asp Glu Gln Phe Pro Ser Ile ProAla 80 85 90 Leu Val His Ser Tyr Met Thr Gly Arg Arg Pro Leu Ser Gln Ala95 100 105 Thr Gly Ala Val Val Ser Arg Pro Val Thr Trp Gln Gly Pro Leu110 115 120 Arg Arg Ser Phe Ser Glu Asp Thr Leu Met Asp Gly Pro Ala Arg125 130 135 Ile Glu Pro Leu Arg Ala Arg Lys Trp Ser Asn Ser Gln Pro Ala140 145 150 Asp Leu Ala His Met Gly Arg Ser Arg Glu Asp Pro Ala Gly Met155 160 165 Glu Ala Ser Thr Met Pro Ile Ser Ala Leu Pro Arg Thr Ser Ser170 175 180 Asp Pro Val Leu Leu Lys Ala Pro Ala Pro Leu Gly Thr Val Ala185 190 195 Asp Ser Leu Arg Ala Ser Asp Gly Gln Leu Gln Ala Lys Ala Pro200 205 210 Thr Lys Pro Pro Arg Thr Pro Ser Phe Glu Leu Pro Asp Ala Ser215 220 225 Glu Arg Pro Pro Thr Tyr Cys Glu Leu Val Pro Arg Val Pro Ser230 235 240 Val Gln Gly Thr Ser Pro Ser Gln Ser Cys Pro Glu Pro Glu Ala245 250 255 Pro Trp Trp Glu Ala Glu Glu Asp Glu Glu Glu Glu Asn Arg Cys260 265 270 Phe Thr Arg Pro Gln Ala Glu Ile Ser Phe Cys Pro His Asp Ala275 280 285 Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg Pro Leu Glu Pro Gln290 295 300 Val Leu His Thr Leu Arg Gly Leu Phe Leu Glu His His Pro Gly305 310 315 Ser Thr Ala Leu His Leu Leu Leu Val Asp Cys Gln Ala Thr Gly320 325 330 Leu Leu Gly Val Thr Arg Asp Gln Arg Gly Asn Met Gly Val Ser335 340 345 Ser Gly Leu Glu Leu Leu Thr Leu Pro His Gly His His Leu Arg350 355 360 Leu Glu Leu Leu Glu Arg His Gln Thr Leu Ala Leu Ala Gly Ala365 370 375 Leu Ala Val Leu Gly Cys Ser Gly Pro Leu Glu Glu Arg Ala Ala380 385 390 Ala Leu Arg Gly Leu Val Glu Leu Ala Leu Ala Leu Arg Pro Gly395 400 405 Ala Ala Gly Asp Leu Pro Gly Leu Ala Ala Val Met Gly Ala Leu410 415 420 Leu Met Pro Gln Val Ser Arg Leu Glu His Thr Trp Arg Gln Leu425 430 435 Arg Arg Ser His Thr Glu Ala Ala Leu Ala Phe Glu Gln Glu Leu440 445 450 Lys Pro Leu Met Arg Ala Leu Asp Glu Gly Ala Gly Pro Cys Asp455 460 465 Pro Gly Glu Val Ala Leu Pro His Val Ala Pro Met Val Arg Leu470 475 480 Leu Glu Gly Glu Glu Val Ala Gly Pro Leu Asp Glu Ser Cys Glu485 490 495 Arg Leu Leu Arg Thr Leu His Gly Ala Arg His Met Val Arg Asp500 505 510 Ala Pro Lys Phe Arg Lys Val Ala Ala Gln Arg Leu Arg Gly Phe515 520 525 Arg Pro Asn Pro Glu Leu Arg Glu Ala Leu Thr Thr Gly Phe Val530 535 540 Arg Arg Leu Leu Trp Gly Ser Arg Gly Ala Gly Ala Pro Arg Ala545 550 555 Glu Arg Phe Glu Lys Phe Gln Arg Val Leu Gly Val Leu Ser Gln560 565 570 Arg Leu Glu Pro Asp Arg 575 576 2 2413 DNA Homo sapiens 2cgggggtgac agcagcccgg agccgcggag cctcagcttc cgcctggacc 50 cagcctcgtgggagccccgc gggtcctgcc cagatgtgga agactgaggc 100 ctgttgaaag tgcagagctcagccctggca ccctctgttc ccaagagctc 150 c atg cag gtg cca cag gat gga gaagac ctt gct ggc 187 Met Gln Val Pro Gln Asp Gly Glu Asp Leu Ala Gly 1 510 caa ccc tgg tac cac ggc ctc ctg tcc cgc cag aag gct 226 Gln Pro TrpTyr His Gly Leu Leu Ser Arg Gln Lys Ala 15 20 25 gaa gct ctt ctt cag caagat ggc gac ttc ctg gtt cgc 265 Glu Ala Leu Leu Gln Gln Asp Gly Asp PheLeu Val Arg 30 35 gcc tct ggg tcc cgt ggg ggc aac ccc gtg atc tcc tgc304 Ala Ser Gly Ser Arg Gly Gly Asn Pro Val Ile Ser Cys 40 45 50 cgc tggcgg ggc tca gcc ctc cat ttt gag gtg ttc cgt 343 Arg Trp Arg Gly Ser AlaLeu His Phe Glu Val Phe Arg 55 60 gtg gcc ctg cgt ccc cgg cca ggc cgaccc aca gcc ctc 382 Val Ala Leu Arg Pro Arg Pro Gly Arg Pro Thr Ala Leu65 70 75 ttt caa ctg gag gat gag caa ttc ccc agc ata ccg gct 421 Phe GlnLeu Glu Asp Glu Gln Phe Pro Ser Ile Pro Ala 80 85 90 ctg gtt cac agt tatatg aca ggc agg cgc cca ctg tcc 460 Leu Val His Ser Tyr Met Thr Gly ArgArg Pro Leu Ser 95 100 cag gcc aca ggg gct gtg gtc tcc agg cct gtg acttgg 499 Gln Ala Thr Gly Ala Val Val Ser Arg Pro Val Thr Trp 105 110 115cag ggg cct ctg cga cgc agc ttt agc gag gac acc ctg 538 Gln Gly Pro LeuArg Arg Ser Phe Ser Glu Asp Thr Leu 120 125 atg gat ggc cca gct cgg atagag cct ctc agg gca agg 577 Met Asp Gly Pro Ala Arg Ile Glu Pro Leu ArgAla Arg 130 135 140 aag tgg agc aac agt cag cct gca gat ttg gca cat atg616 Lys Trp Ser Asn Ser Gln Pro Ala Asp Leu Ala His Met 145 150 155 gggcgg tca aga gaa gac ccc gct ggg atg gaa gcc tcc 655 Gly Arg Ser Arg GluAsp Pro Ala Gly Met Glu Ala Ser 160 165 acc atg ccc ata tct gcc ttg ccccga acg agc agt gac 694 Thr Met Pro Ile Ser Ala Leu Pro Arg Thr Ser SerAsp 170 175 180 ccg gtg ttg ctg aag gcc cct gct ccc ctg gga act gtt 733Pro Val Leu Leu Lys Ala Pro Ala Pro Leu Gly Thr Val 185 190 gcc gac agtctc agg gcc tcc gat ggg cag ctt caa gcc 772 Ala Asp Ser Leu Arg Ala SerAsp Gly Gln Leu Gln Ala 195 200 205 aag gca cca acg aag ccc ccc cgg acaccc tcc ttc gaa 811 Lys Ala Pro Thr Lys Pro Pro Arg Thr Pro Ser Phe Glu210 215 220 ctg cct gat gcc tct gaa cgt ccc ccg acg tac tgc gag 850 LeuPro Asp Ala Ser Glu Arg Pro Pro Thr Tyr Cys Glu 225 230 ctg gtg ccc cgagtg ccc agt gtc cag gga aca tcc ccg 889 Leu Val Pro Arg Val Pro Ser ValGln Gly Thr Ser Pro 235 240 245 agc caa agc tgc cca gag cca gag gcc ccatgg tgg gag 928 Ser Gln Ser Cys Pro Glu Pro Glu Ala Pro Trp Trp Glu 250255 gcc gag gag gat gag gag gaa gag aat aga tgt ttt aca 967 Ala Glu GluAsp Glu Glu Glu Glu Asn Arg Cys Phe Thr 260 265 270 aga cca cag gct gagatc tct ttc tgc ccc cat gat gcc 1006 Arg Pro Gln Ala Glu Ile Ser Phe CysPro His Asp Ala 275 280 285 ccc tcc tgc ctg ctg ggc ccc cag aat cgg cccctg gaa 1045 Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg Pro Leu Glu 290 295ccc caa gtc ctg cat acc ctc cgt ggc ctg ttc ctg gag 1084 Pro Gln Val LeuHis Thr Leu Arg Gly Leu Phe Leu Glu 300 305 310 cac cat cct ggg agc accgcc ctt cac ctg cta ttg gta 1123 His His Pro Gly Ser Thr Ala Leu His LeuLeu Leu Val 315 320 gac tgc cag gcc aca ggc ctc ctg gga gtg acc aga gat1162 Asp Cys Gln Ala Thr Gly Leu Leu Gly Val Thr Arg Asp 325 330 335 cagcgg ggc aac atg gga gtc tca tct ggc ctg gag ctg 1201 Gln Arg Gly Asn MetGly Val Ser Ser Gly Leu Glu Leu 340 345 350 ctc act ctt ccc cat gga caccac ttg agg ttg gaa ctg 1240 Leu Thr Leu Pro His Gly His His Leu Arg LeuGlu Leu 355 360 ctg gag agg cat cag aca ctg gcg ctg gcc ggg gcg ctg 1279Leu Glu Arg His Gln Thr Leu Ala Leu Ala Gly Ala Leu 365 370 375 gcg gtgctg ggc tgc tcg ggg ccg ctg gag gag cgc gca 1318 Ala Val Leu Gly Cys SerGly Pro Leu Glu Glu Arg Ala 380 385 gcc gca ctg agg gga ctg gta gag ctggcg ctg gcg ctg 1357 Ala Ala Leu Arg Gly Leu Val Glu Leu Ala Leu Ala Leu390 395 400 cgg cca ggg gcg gcg ggg gac ctg ccc ggg ctg gct gca 1396 ArgPro Gly Ala Ala Gly Asp Leu Pro Gly Leu Ala Ala 405 410 415 gtc atg ggcgcc ctg ctc atg ccc cag gtg tcc cgg ttg 1435 Val Met Gly Ala Leu Leu MetPro Gln Val Ser Arg Leu 420 425 gag cac acg tgg cgc cag ctc cga agg agccac acg gag 1474 Glu His Thr Trp Arg Gln Leu Arg Arg Ser His Thr Glu 430435 440 gct gcg ctg gcc ttt gag cag gag ctg aag ccg ctg atg 1513 Ala AlaLeu Ala Phe Glu Gln Glu Leu Lys Pro Leu Met 445 450 cgg gct ctg gat gagggc gct gga ccc tgc gac ccc ggc 1552 Arg Ala Leu Asp Glu Gly Ala Gly ProCys Asp Pro Gly 455 460 465 gag gtg gcg ctg ccg cac gtg gca ccc atg gttcgc cta 1591 Glu Val Ala Leu Pro His Val Ala Pro Met Val Arg Leu 470 475480 ctg gag ggc gag gaa gtc gcg ggg ccg ctg gac gag agc 1630 Leu Glu GlyGlu Glu Val Ala Gly Pro Leu Asp Glu Ser 485 490 tgt gag cgg ctg ttg cgcacc ctg cac ggg gcg cgt cac 1669 Cys Glu Arg Leu Leu Arg Thr Leu His GlyAla Arg His 495 500 505 atg gtc cgg gac gca ccc aaa ttc cgc aag gtg gcagcc 1708 Met Val Arg Asp Ala Pro Lys Phe Arg Lys Val Ala Ala 510 515 cagcgc ctg cga gga ttc cgg cct aac ccg gag ctg agg 1747 Gln Arg Leu Arg GlyPhe Arg Pro Asn Pro Glu Leu Arg 520 525 530 gag gcc ctg acc acc ggc ttcgtg cgg agg ctg ctc tgg 1786 Glu Ala Leu Thr Thr Gly Phe Val Arg Arg LeuLeu Trp 535 540 545 ggt agc cgg ggc gcg gga gct ccg cgc gct gaa cgc ttt1825 Gly Ser Arg Gly Ala Gly Ala Pro Arg Ala Glu Arg Phe 550 555 gag aagttc cag cgc gtc ctc ggc gtc ctg tcg cag cgc 1864 Glu Lys Phe Gln Arg ValLeu Gly Val Leu Ser Gln Arg 560 565 570 ctg gag cct gac cgc t gagagcgcagacacccttct tcacacccgg 1910 Leu Glu Pro Asp Arg 575 576 gacccccaggtttttgcgaa ccccagaaga gaccaaagga gtcgtcccag 1960 gctcctcgcg cctcaggtggaatcctgccc tgtgcctcac agaagaggtg 2010 gggaccgcag tcagggtcac ctggaccatggtgaacatgt gacctgcaga 2060 tctggcatca gaggccagag ttcaaatgtg actccacctcttaaaagccg 2110 tgatttctag cagttgactt cacctctgtg tcggccttta acaaaatcat2160 agccatacag cagctcaggc ctgtaatctc agcactttgg gaggccgagg 2210cggaaggaag gcttgaggcc aggagttcaa gaccagccag ggcaacatgg 2260 tgagacctcatctctacaaa aactgaaaaa taaaaaactt ttaaaaaatg 2310 taaaaaaaaa aaaaaaagggcggccgcgac tctagagtcg acctgcagaa 2360 gcttggccgc catggcccaa cttgtttattgcagcttata atggttacaa 2410 ata 2413 3 501 PRT Homo sapiens 3 Met Gln AspArg Arg Ala Leu Ser Leu Lys Ala His Gln Ser Glu 1 5 10 15 Ser Tyr LeuPro Ile Gly Cys Lys Leu Pro Pro Gln Ser Ser Gly 20 25 30 Val Asp Thr SerPro Cys Pro Asn Ser Pro Val Phe Arg Thr Gly 35 40 45 Ser Glu Pro Ala LeuSer Pro Ala Val Val Arg Arg Val Ser Ser 50 55 60 Asp Ala Arg Ala Gly GluAla Leu Arg Gly Ser Asp Ser Gln Leu 65 70 75 Cys Pro Lys Pro Pro Pro LysPro Cys Lys Val Pro Phe Leu Lys 80 85 90 Val Pro Ser Ser Pro Ser Ala TrpLeu Asn Ser Glu Ala Asn Tyr 95 100 105 Cys Glu Leu Asn Pro Ala Phe AlaThr Gly Cys Gly Arg Gly Ala 110 115 120 Lys Leu Pro Ser Cys Ala Gln GlySer His Thr Glu Leu Leu Thr 125 130 135 Ala Lys Gln Asn Glu Ala Pro GlyPro Arg Asn Ser Gly Val Asn 140 145 150 Tyr Leu Ile Leu Asp Asp Asp AspArg Glu Arg Pro Trp Glu Pro 155 160 165 Ala Ala Ala Gln Met Glu Lys GlyGln Trp Asp Lys Gly Glu Phe 170 175 180 Val Thr Pro Leu Leu Glu Thr ValSer Ser Phe Arg Pro Asn Glu 185 190 195 Phe Glu Ser Lys Phe Leu Pro ProGlu Asn Lys Pro Leu Glu Thr 200 205 210 Ala Met Leu Lys Arg Ala Lys GluLeu Phe Thr Asn Asn Asp Pro 215 220 225 Lys Val Ile Ala Gln His Val LeuSer Met Asp Cys Arg Val Ala 230 235 240 Arg Ile Leu Gly Val Ser Glu GluMet Arg Arg Asn Met Gly Val 245 250 255 Ser Ser Gly Leu Glu Leu Ile ThrLeu Pro His Gly His Gln Leu 260 265 270 Arg Leu Asp Ile Ile Glu Arg HisAsn Thr Met Ala Ile Gly Ile 275 280 285 Ala Val Asp Ile Leu Gly Cys ThrGly Thr Leu Glu Asp Arg Ala 290 295 300 Ala Thr Leu Ser Lys Ile Ile GlnVal Ala Val Glu Leu Lys Asp 305 310 315 Ser Met Gly Asp Leu Tyr Ser PheSer Ala Leu Met Lys Ala Leu 320 325 330 Glu Met Pro Gln Ile Thr Arg LeuGlu Lys Thr Trp Thr Ala Leu 335 340 345 Arg His Gln Tyr Thr Gln Thr AlaIle Leu Tyr Glu Lys Gln Leu 350 355 360 Lys Pro Phe Ser Lys Leu Leu HisGlu Gly Arg Glu Ser Thr Cys 365 370 375 Val Pro Pro Asn Asn Val Ser ValPro Leu Leu Met Pro Leu Val 380 385 390 Thr Leu Met Glu Arg Gln Ala ValThr Phe Glu Gly Thr Asp Met 395 400 405 Trp Glu Lys Asn Asp Gln Ser CysGlu Ile Met Leu Asn His Leu 410 415 420 Ala Thr Ala Arg Phe Met Ala GluAla Ala Asp Ser Tyr Arg Met 425 430 435 Asn Ala Glu Arg Ile Leu Ala GlyPhe Gln Pro Asp Glu Glu Met 440 445 450 Asn Glu Ile Cys Lys Thr Glu PheGln Met Arg Leu Leu Trp Gly 455 460 465 Ser Lys Gly Ala Gln Val Asn GlnThr Glu Arg Tyr Glu Lys Phe 470 475 480 Asn Gln Ile Leu Thr Ala Leu SerArg Lys Leu Glu Pro Pro Pro 485 490 495 Val Lys Gln Ala Glu Leu 500 5014 2174 DNA Homo sapiens 4 ggcccctgga gtccagccgc agtggtcact gcttaaatatcacttctcgg 50 gagatatttc cttttgtaat ttgccctcgg tcttgtctta tcttcgaaag 100gttgctggaa tttctctgtt ccttggagtt tgggggtttt ttgatttgtt 150 ttttctttggtgcttgtaaa gaaacaaaga aaagagtggt agccagcccg 200 cctgcctgga tcac atg caggac aga aga gcc ttg tcc ctc 241 Met Gln Asp Arg Arg Ala Leu Ser Leu 1 5aaa gcc cac cag tca gag agc tac ctg ccg att ggc tgc 280 Lys Ala His GlnSer Glu Ser Tyr Leu Pro Ile Gly Cys 10 15 20 aag ctg cca cct cag tcc tcgggt gtg gac aca agc ccc 319 Lys Leu Pro Pro Gln Ser Ser Gly Val Asp ThrSer Pro 25 30 35 tgc cca aac tca cct gtg ttc agg acg gga agc gag cct 358Cys Pro Asn Ser Pro Val Phe Arg Thr Gly Ser Glu Pro 40 45 gcc ctg agccca gca gtg gtt cgg agg gtc tcc tca gac 397 Ala Leu Ser Pro Ala Val ValArg Arg Val Ser Ser Asp 50 55 60 gcc agg gct ggg gag gcg ctg agg gga tcagac agt caa 436 Ala Arg Ala Gly Glu Ala Leu Arg Gly Ser Asp Ser Gln 6570 ctg tgc cct aag ccc ccg cct aag ccc tgc aag gtg ccg 475 Leu Cys ProLys Pro Pro Pro Lys Pro Cys Lys Val Pro 75 80 85 ttc ctc aag gtt ccc tcgtct ccc tct gcc tgg ctc aac 514 Phe Leu Lys Val Pro Ser Ser Pro Ser AlaTrp Leu Asn 90 95 100 tca gag gcc aac tac tgt gaa ctg aac cca gcg tttgcc 553 Ser Glu Ala Asn Tyr Cys Glu Leu Asn Pro Ala Phe Ala 105 110 acaggc tgc ggc agg gga gca aag cta ccc tca tgt gcc 592 Thr Gly Cys Gly ArgGly Ala Lys Leu Pro Ser Cys Ala 115 120 125 cag gga agc cac aca gaa ctgctc aca gcc aag cag aat 631 Gln Gly Ser His Thr Glu Leu Leu Thr Ala LysGln Asn 130 135 gag gcg cca ggt ccc cgg aac tct ggc gtc aac tac ttg 670Glu Ala Pro Gly Pro Arg Asn Ser Gly Val Asn Tyr Leu 140 145 150 atc cttgat gat gat gac agg gaa aga cct tgg gaa cct 709 Ile Leu Asp Asp Asp AspArg Glu Arg Pro Trp Glu Pro 155 160 165 gcg gca gct cag atg gag aag gggcag tgg gac aag ggc 748 Ala Ala Ala Gln Met Glu Lys Gly Gln Trp Asp LysGly 170 175 gag ttt gtg acg ccc ctc ctg gag act gtc tcc tcc ttc 787 GluPhe Val Thr Pro Leu Leu Glu Thr Val Ser Ser Phe 180 185 190 agg ccc aacgag ttt gag tca aag ttc ctt ccc cct gag 826 Arg Pro Asn Glu Phe Glu SerLys Phe Leu Pro Pro Glu 195 200 aat aag ccc ctg gaa aca gca atg ttg aaacgt gca aaa 865 Asn Lys Pro Leu Glu Thr Ala Met Leu Lys Arg Ala Lys 205210 215 gaa ctg ttc acc aac aac gac ccc aag gtc atc gcc cag 904 Glu LeuPhe Thr Asn Asn Asp Pro Lys Val Ile Ala Gln 220 225 230 cac gta ctg agcatg gac tgc agg gtt gct agg ata ctt 943 His Val Leu Ser Met Asp Cys ArgVal Ala Arg Ile Leu 235 240 gga gtc tct gaa gag atg agg agg aac atg ggggtg agc 982 Gly Val Ser Glu Glu Met Arg Arg Asn Met Gly Val Ser 245 250255 tca ggc ctg gaa ctc att acc ttg cct cac gga cac cag 1021 Ser Gly LeuGlu Leu Ile Thr Leu Pro His Gly His Gln 260 265 ctg cgc ctg gac ata attgaa aga cac aac aca atg gcc 1060 Leu Arg Leu Asp Ile Ile Glu Arg His AsnThr Met Ala 270 275 280 atc ggc att gca gtg gac att ctg gga tgc acg ggcact 1099 Ile Gly Ile Ala Val Asp Ile Leu Gly Cys Thr Gly Thr 285 290 295ttg gag gac cga gcg gcc act ctg agt aag atc atc cag 1138 Leu Glu Asp ArgAla Ala Thr Leu Ser Lys Ile Ile Gln 300 305 gtg gcg gtg gaa ctg aag gattcc atg ggg gac ctc tat 1177 Val Ala Val Glu Leu Lys Asp Ser Met Gly AspLeu Tyr 310 315 320 tcc ttc tca gct ctc atg aaa gcc ctg gaa atg cca cag1216 Ser Phe Ser Ala Leu Met Lys Ala Leu Glu Met Pro Gln 325 330 atc acaagg tta gaa aag acg tgg act gct ctg cgg cac 1255 Ile Thr Arg Leu Glu LysThr Trp Thr Ala Leu Arg His 335 340 345 cag tac acc caa act gcc att ctctat gag aaa cag ctg 1294 Gln Tyr Thr Gln Thr Ala Ile Leu Tyr Glu Lys GlnLeu 350 355 360 aag ccc ttc agc aaa ctc ctg cat gaa ggc aga gag tcc 1333Lys Pro Phe Ser Lys Leu Leu His Glu Gly Arg Glu Ser 365 370 aca tgt gttccc cca aac aat gta tca gtc cca ctg ctg 1372 Thr Cys Val Pro Pro Asn AsnVal Ser Val Pro Leu Leu 375 380 385 atg ccg ctt gtg acg tta atg gag cgccag gct gtg act 1411 Met Pro Leu Val Thr Leu Met Glu Arg Gln Ala Val Thr390 395 ttt gaa gga acc gac atg tgg gaa aaa aac gac cag agc 1450 Phe GluGly Thr Asp Met Trp Glu Lys Asn Asp Gln Ser 400 405 410 tgt gaa atc atgctg aac cat ttg gca aca gcg cga ttc 1489 Cys Glu Ile Met Leu Asn His LeuAla Thr Ala Arg Phe 415 420 425 atg gcc gag gct gca gac agc tac cgg atgaat gct gag 1528 Met Ala Glu Ala Ala Asp Ser Tyr Arg Met Asn Ala Glu 430435 agg atc ctg gca ggt ttt caa cca gat gaa gaa atg aat 1567 Arg Ile LeuAla Gly Phe Gln Pro Asp Glu Glu Met Asn 440 445 450 gaa atc tgc aag actgaa ttt caa atg cga ttg cta tgg 1606 Glu Ile Cys Lys Thr Glu Phe Gln MetArg Leu Leu Trp 455 460 ggc agc aaa ggt gca caa gtc aat cag aca gag agatat 1645 Gly Ser Lys Gly Ala Gln Val Asn Gln Thr Glu Arg Tyr 465 470 475gag aaa ttc aac cag att tta act gcc ctc tcg cgt aaa 1684 Glu Lys Phe AsnGln Ile Leu Thr Ala Leu Ser Arg Lys 480 485 490 ttg gaa cct cct cct gtaaag cag gca gag ctt tga 1720 Leu Glu Pro Pro Pro Val Lys Gln Ala Glu Leu495 500 501 taactctcca gagaaccttt agaatatctt ttcaagtttc cccagcttca 1770tctttgggaa agcttactgt ttttgataaa gtaataatgt gcaaatctga 1820 caatatacaagcttttagta tccacaggat attaaacgtg taaattgcac 1870 agagcacact tatttatgaattgtctaaag ttactactga ttttaaaatg 1920 aataatttat tattaaggta actactgctaatgttgatca gcaaatttaa 1970 gagaagacct agctatgttg gctggttgct ttctattatcatggtatttg 2020 accattttag ttttaattcc atgtcagata agtgtaaata gaagagttta2070 aaagcatgaa acatttcaga aggtatcagt tatatgatat tctttaaaca 2120aatatgaaaa atgtaaatac tcatgaatga aaatacatct ttttgtgaaa 2170 cagt 2174 5703 PRT Homo sapiens 5 Met Thr Ala Val Gly Arg Arg Cys Pro Ala Leu GlySer Arg Gly 1 5 10 15 Ala Ala Gly Glu Pro Glu Ala Gly Ser Asp Tyr ValLys Phe Ser 20 25 30 Lys Glu Lys Tyr Ile Leu Asp Ser Ser Pro Glu Lys LeuHis Lys 35 40 45 Glu Leu Glu Glu Glu Leu Lys Leu Ser Ser Thr Asp Leu ArgSer 50 55 60 His Ala Trp Tyr His Gly Arg Ile Pro Arg Glu Val Ser Glu Thr65 70 75 Leu Val Gln Arg Asn Gly Asp Phe Leu Ile Arg Asp Ser Leu Thr 8085 90 Ser Leu Gly Asp Tyr Val Leu Thr Cys Arg Trp Arg Asn Gln Ala 95 100105 Leu His Phe Lys Ile Asn Lys Val Val Val Lys Ala Gly Glu Ser 110 115120 Tyr Thr His Ile Gln Tyr Leu Phe Glu Gln Glu Ser Phe Asp His 125 130135 Val Pro Ala Leu Val Arg Tyr His Val Gly Ser Arg Lys Ala Val 140 145150 Ser Glu Gln Ser Gly Ala Ile Ile Tyr Cys Pro Val Asn Arg Thr 155 160165 Phe Pro Leu Arg Tyr Leu Glu Ala Ser Tyr Gly Leu Gly Gln Gly 170 175180 Ser Ser Lys Pro Ala Ser Pro Val Ser Pro Ser Gly Pro Lys Gly 185 190195 Ser His Met Lys Arg Arg Ser Val Thr Met Thr Asp Gly Leu Thr 200 205210 Ala Asp Lys Val Thr Arg Ser Asp Gly Cys Pro Thr Ser Thr Ser 215 220225 Leu Pro Arg Pro Arg Asp Ser Ile Arg Ser Cys Ala Leu Ser Met 230 235240 Asp Gln Ile Pro Asp Leu His Ser Pro Met Ser Pro Ile Ser Glu 245 250255 Ser Pro Ser Ser Pro Ala Tyr Ser Thr Val Thr Arg Val His Ala 260 265270 Ala Pro Ala Ala Pro Ser Ala Thr Ala Leu Pro Ala Ser Pro Val 275 280285 Ala Arg Cys Ser Ser Glu Pro Gln Leu Cys Pro Gly Ser Ala Pro 290 295300 Lys Thr His Gly Glu Ser Asp Lys Gly Pro His Thr Ser Pro Ser 305 310315 His Thr Leu Gly Lys Ala Ser Pro Ser Pro Ser Leu Ser Ser Tyr 320 325330 Ser Asp Pro Asp Ser Gly His Tyr Cys Gln Leu Gln Pro Pro Val 335 340345 Arg Gly Ser Arg Glu Trp Ala Ala Thr Glu Thr Ser Ser Gln Gln 350 355360 Ala Arg Ser Tyr Gly Glu Arg Leu Lys Glu Leu Ser Glu Asn Gly 365 370375 Ala Pro Glu Gly Asp Trp Gly Lys Thr Phe Thr Val Pro Ile Val 380 385390 Glu Val Thr Ser Ser Phe Asn Pro Ala Thr Phe Gln Ser Leu Leu 395 400405 Ile Pro Arg Asp Asn Arg Pro Leu Glu Val Gly Leu Leu Arg Lys 410 415420 Val Lys Glu Leu Leu Ala Glu Val Asp Ala Arg Thr Leu Ala Arg 425 430435 His Val Thr Lys Val Asp Cys Leu Val Ala Arg Ile Leu Gly Val 440 445450 Thr Lys Glu Met Gln Thr Leu Met Gly Val Arg Trp Gly Met Glu 455 460465 Leu Leu Thr Leu Pro His Gly Arg Gln Leu Arg Leu Asp Leu Leu 470 475480 Glu Arg Phe His Thr Met Ser Ile Met Leu Ala Val Asp Ile Leu 485 490495 Gly Cys Thr Gly Ser Ala Glu Glu Arg Ala Ala Leu Leu His Lys 500 505510 Thr Ile Gln Leu Ala Ala Glu Leu Arg Gly Thr Met Gly Asn Met 515 520525 Phe Ser Phe Ala Ala Val Met Gly Ala Leu Asp Met Ala Gln Ile 530 535540 Ser Arg Leu Glu Gln Thr Trp Val Thr Leu Arg Gln Arg His Thr 545 550555 Glu Gly Ala Ile Leu Tyr Glu Lys Lys Leu Lys Pro Phe Leu Lys 560 565570 Ser Leu Asn Glu Gly Lys Glu Gly Pro Pro Leu Ser Asn Thr Thr 575 580585 Phe Pro His Val Leu Pro Leu Ile Thr Leu Leu Glu Cys Asp Ser 590 595600 Ala Pro Pro Glu Gly Pro Glu Pro Trp Gly Ser Thr Glu His Gly 605 610615 Val Glu Val Val Leu Ala His Leu Glu Ala Ala Arg Thr Val Ala 620 625630 His His Gly Gly Leu Tyr His Thr Asn Ala Glu Val Lys Leu Gln 635 640645 Gly Phe Gln Ala Arg Pro Glu Leu Leu Glu Val Phe Ser Thr Glu 650 655660 Phe Gln Met Arg Leu Leu Trp Gly Ser Gln Gly Ala Ser Ser Ser 665 670675 Gln Ala Arg Arg Tyr Glu Lys Phe Asp Lys Val Leu Thr Ala Leu 680 685690 Ser His Lys Leu Glu Pro Ala Val Arg Ser Ser Glu Leu 695 700 703 62153 DNA Homo sapiens 6 taggaggtcc ccgggttgcc ggcggcgaca gcgggggaag catg 44 Met 1 act gct gtg ggc cga agg tgc ccc gcg ctg ggg tcc cga 83 ThrAla Val Gly Arg Arg Cys Pro Ala Leu Gly Ser Arg 5 10 ggg gct gct gga gagcca gag gct ggc agc gac tat gtg 122 Gly Ala Ala Gly Glu Pro Glu Ala GlySer Asp Tyr Val 15 20 25 aag ttc tcc aag gag aag tac atc ctg gac tca tcgcca 161 Lys Phe Ser Lys Glu Lys Tyr Ile Leu Asp Ser Ser Pro 30 35 40 gagaaa ctc cac aag gaa ttg gag gag gag ctc aaa ctc 200 Glu Lys Leu His LysGlu Leu Glu Glu Glu Leu Lys Leu 45 50 agc agc acg gat ctc cgc agc catgcc tgg tac cat ggc 239 Ser Ser Thr Asp Leu Arg Ser His Ala Trp Tyr HisGly 55 60 65 cgc atc ccc cga gag gtc tcg gag acc ttg gta caa cgc 278 ArgIle Pro Arg Glu Val Ser Glu Thr Leu Val Gln Arg 70 75 aac ggc gac ttcctc atc cgg gac tcg ctc acc agc ctg 317 Asn Gly Asp Phe Leu Ile Arg AspSer Leu Thr Ser Leu 80 85 90 ggc gac tat gtg ctc acg tgc cgc tgg cgc aaccag gcc 356 Gly Asp Tyr Val Leu Thr Cys Arg Trp Arg Asn Gln Ala 95 100105 ttg cac ttc aag atc aac aag gtg gtg gtg aag gca ggc 395 Leu His PheLys Ile Asn Lys Val Val Val Lys Ala Gly 110 115 gag agc tac aca cac atccag tac ctg ttt gag cag gag 434 Glu Ser Tyr Thr His Ile Gln Tyr Leu PheGlu Gln Glu 120 125 130 agc ttt gac cac gtg ccc gcc ctc gtg cgc tat catgtg 473 Ser Phe Asp His Val Pro Ala Leu Val Arg Tyr His Val 135 140 ggcagc cgc aag gct gtg tca gag cag agt ggt gcc atc 512 Gly Ser Arg Lys AlaVal Ser Glu Gln Ser Gly Ala Ile 145 150 155 atc tac tgc ccg gtg aac cgcacc ttc cca ctg cgc tac 551 Ile Tyr Cys Pro Val Asn Arg Thr Phe Pro LeuArg Tyr 160 165 170 ctc gag gcc agc tat ggc ctg gga cag ggg agt agc aag590 Leu Glu Ala Ser Tyr Gly Leu Gly Gln Gly Ser Ser Lys 175 180 cct gctagc ccc gtc agc ccc tca ggc ccc aag ggc agc 629 Pro Ala Ser Pro Val SerPro Ser Gly Pro Lys Gly Ser 185 190 195 cac atg aag cgg cgc agc gtc accatg acc gat ggg ctc 668 His Met Lys Arg Arg Ser Val Thr Met Thr Asp GlyLeu 200 205 act gct gac aag gtc acc cgc agc gat ggc tgc ccc acc 707 ThrAla Asp Lys Val Thr Arg Ser Asp Gly Cys Pro Thr 210 215 220 agt acg tcgctg ccc cgc cct cgg gac tcc atc cgc agc 746 Ser Thr Ser Leu Pro Arg ProArg Asp Ser Ile Arg Ser 225 230 235 tgt gcc ctc agc atg gac cag atc ccagac ctg cac tca 785 Cys Ala Leu Ser Met Asp Gln Ile Pro Asp Leu His Ser240 245 ccc atg tcg ccc atc tcc gag agc cct agc tcc cct gcc 824 Pro MetSer Pro Ile Ser Glu Ser Pro Ser Ser Pro Ala 250 255 260 tac agc act gtaacc cgt gtc cat gcc gcc cct gca gcc 863 Tyr Ser Thr Val Thr Arg Val HisAla Ala Pro Ala Ala 265 270 cct tct gcc aca gca ttg cct gcc tcc cct gtcgcc cgc 902 Pro Ser Ala Thr Ala Leu Pro Ala Ser Pro Val Ala Arg 275 280285 tgt tcc agt gag ccc cag ctg tgt ccc gga agt gcc cca 941 Cys Ser SerGlu Pro Gln Leu Cys Pro Gly Ser Ala Pro 290 295 300 aag acc cat ggg gagtca gac aag ggc ccc cac acc agc 980 Lys Thr His Gly Glu Ser Asp Lys GlyPro His Thr Ser 305 310 ccc tcc cac acc ctt ggc aag gcc tcc ccg tca ccatca 1019 Pro Ser His Thr Leu Gly Lys Ala Ser Pro Ser Pro Ser 315 320 325ctc agc agc tac agt gac ccg gac tct ggc cac tac tgc 1058 Leu Ser Ser TyrSer Asp Pro Asp Ser Gly His Tyr Cys 330 335 cag ctc cag cct ccc gtg cgtggc agc cga gag tgg gca 1097 Gln Leu Gln Pro Pro Val Arg Gly Ser Arg GluTrp Ala 340 345 350 gcg act gag acc tcc agc cag cag gcc agg agc tat ggg1136 Ala Thr Glu Thr Ser Ser Gln Gln Ala Arg Ser Tyr Gly 355 360 365 gagagg cta aag gaa ctg tca gaa aat ggg gcc cct gaa 1175 Glu Arg Leu Lys GluLeu Ser Glu Asn Gly Ala Pro Glu 370 375 ggg gac tgg ggc aag acc ttc acagtc ccc atc gtg gaa 1214 Gly Asp Trp Gly Lys Thr Phe Thr Val Pro Ile ValGlu 380 385 390 gtc act tct tcc ttc aac ccg gcc acc ttc cag tca cta 1253Val Thr Ser Ser Phe Asn Pro Ala Thr Phe Gln Ser Leu 395 400 ctg atc cccagg gat aac cgg cca ctg gag gtg ggc ctt 1292 Leu Ile Pro Arg Asp Asn ArgPro Leu Glu Val Gly Leu 405 410 415 ctg cgc aag gtc aag gag ctg ctg gcagaa gtg gat gcc 1331 Leu Arg Lys Val Lys Glu Leu Leu Ala Glu Val Asp Ala420 425 430 cgg acg ctg gcc cgg cat gtc acc aag gtg gac tgc ctg 1370 ArgThr Leu Ala Arg His Val Thr Lys Val Asp Cys Leu 435 440 gtt gct agg atactg ggc gtt acc aag gag atg cag acc 1409 Val Ala Arg Ile Leu Gly Val ThrLys Glu Met Gln Thr 445 450 455 cta atg gga gtc cgc tgg ggc atg gaa ctgctc acc ctc 1448 Leu Met Gly Val Arg Trp Gly Met Glu Leu Leu Thr Leu 460465 ccc cat ggc cgg cag cta cgc cta gac ctg ctg gaa agg 1487 Pro His GlyArg Gln Leu Arg Leu Asp Leu Leu Glu Arg 470 475 480 ttc cac acc atg tccatc atg ctg gcc gtg gac atc ctg 1526 Phe His Thr Met Ser Ile Met Leu AlaVal Asp Ile Leu 485 490 495 ggc tgc acc ggc tct gcg gag gag cgg gca gcgctg ctg 1565 Gly Cys Thr Gly Ser Ala Glu Glu Arg Ala Ala Leu Leu 500 505cac aag acc att cag ctg gcg gcc gag cta cgg ggg act 1604 His Lys Thr IleGln Leu Ala Ala Glu Leu Arg Gly Thr 510 515 520 atg ggc aac atg ttc agcttc gcg gcg gtc atg ggt gcc 1643 Met Gly Asn Met Phe Ser Phe Ala Ala ValMet Gly Ala 525 530 ctg gac atg gct cag att tct cgg ctg gag cag aca tgg1682 Leu Asp Met Ala Gln Ile Ser Arg Leu Glu Gln Thr Trp 535 540 545 gtgacc ctg cgg cag cga cac aca gag ggt gcc atc ctg 1721 Val Thr Leu Arg GlnArg His Thr Glu Gly Ala Ile Leu 550 555 560 tac gag aag aag ctc aag cctttt ctc aag agc ctc aac 1760 Tyr Glu Lys Lys Leu Lys Pro Phe Leu Lys SerLeu Asn 565 570 gag ggc aaa gaa ggc ccg ccg ctg agc aac acc acg ttt 1799Glu Gly Lys Glu Gly Pro Pro Leu Ser Asn Thr Thr Phe 575 580 585 cct catgtg ctg ccc ctc atc acc ctg ctg gag tgt gac 1838 Pro His Val Leu Pro LeuIle Thr Leu Leu Glu Cys Asp 590 595 tcg gcc cca cca gag ggc cct gag ccctgg ggc agc acg 1877 Ser Ala Pro Pro Glu Gly Pro Glu Pro Trp Gly Ser Thr600 605 610 gag cac ggc gtg gag gtg gtg ctg gct cac ctg gag gcc 1916 GluHis Gly Val Glu Val Val Leu Ala His Leu Glu Ala 615 620 625 gcc cgc acagtg gca cac cac gga ggc ctg tac cac acc 1955 Ala Arg Thr Val Ala His HisGly Gly Leu Tyr His Thr 630 635 aat gct gaa gtc aag ctg cag ggg ttc caggcc cgg ccg 1994 Asn Ala Glu Val Lys Leu Gln Gly Phe Gln Ala Arg Pro 640645 650 gag ctc ctg gag gtg ttc agc acg gag ttc cag atg cgc 2033 Glu LeuLeu Glu Val Phe Ser Thr Glu Phe Gln Met Arg 655 660 ctt ctc tgg ggc agtcag ggt gcc agc agc agc cag gcc 2072 Leu Leu Trp Gly Ser Gln Gly Ala SerSer Ser Gln Ala 665 670 675 cgg cgc tat gag aag ttc gac aag gtc ctc actgcc ctg 2111 Arg Arg Tyr Glu Lys Phe Asp Lys Val Leu Thr Ala Leu 680 685690 tcc cac aag ctg gaa cct gct gtc cgc tcc agc gag ctg 2150 Ser His LysLeu Glu Pro Ala Val Arg Ser Ser Glu Leu 695 700 703 tga 2153 7 30 DNAArtificial Sequence artificial sequence 7 actgaggcct gttgaaagtgcagagctcag 30 8 18 DNA Artificial Sequence artificial sequence 8gctgaagaag agcttcag 18 9 48 DNA Artificial Sequence artificial sequence9 caatgccgat ggccattgtg ttgtgtcttt caattatgtc caggcgca 48 10 18 DNAArtificial Sequence artificial sequence 10 atcccagaat gtccactg 18 11 50DNA Artificial Sequence artificial sequence 11 ggccagcatg atggacatggtgtggaacct ttccagcagg tctaggcgta 50 12 18 DNA Artificial Sequenceartificial sequence 12 ggtgcagccc aggatgtc 18 13 254 DNA unknown unknownsource 1-254 13 gtggagggcg ggggtgacag cagcccggag ccgcggagcc tcagcttccg50 cctggaccca gcctcgtggg agccccgcgg gtcctgccca gatgtggaag 100 actgaggcctgttgaaagtg cagagctcag ccctggcacc ctctgttccc 150 aagagctcca tgcaggtgccacaggatgga gaagaccttg ctggccaacc 200 ttggtaccac ggcctcctgt cccgccagaaggctgaagct cttcttcagc 250 aaaa 254 14 212 DNA unknown unknown source1-212 14 catcgcccag cacgtactga gcatggactg cagggttgct aggatacttg 50gagtctctna agagatgagg aggaacatgg gggtgagctc aggcctggaa 100 ctcattaccttgcctcacgg acaccagctg cgcctggaca taattgaaag 150 acacaacaca atggccatcggcattgcagt ggacattctg ggatgcacgg 200 gcactttgga gg 212 15 242 DNAunknown source unknown source 1-242 15 gctggcagaa gtggatgccc ggacgctggcccggcatgtc accaaggtgg 50 actgcctggt tgctaggata ctgggcgtta ccaaggagatgcagacccta 100 atgggagtcc gctggggcat ggaactgctc accctccccc atggccggca150 gctacgccta gacctgctgg aaaggttcca caccatgtcc atcatgctgg 200ccgnggacat cctgggctgc accggctctg cggaggagcg gg 242 16 99 PRT Homosapiens 16 Glu Gln Leu Arg Gly Glu Pro Trp Phe His Gly Lys Leu Ser Arg 15 10 15 Arg Glu Ala Glu Ala Leu Leu Gln Leu Asn Gly Asp Phe Leu Val 2025 30 Arg Glu Ser Thr Thr Thr Pro Gly Gln Tyr Val Gly Leu Gln Ser 35 4045 Gly Gln Pro Lys His Leu Leu Leu Val Asp Pro Glu Gly Val Val 50 55 60Arg Thr Lys Asp His Arg Phe Glu Ser Val Ser His Leu Ile Ser 65 70 75 TyrHis Met Asp Asn Pro Ile Ile Ser Ala Gly Ser Glu Leu Cys 80 85 90 Leu GlnGln Pro Val Glu Arg Lys Leu 95 99 17 99 PRT Homo sapiens 17 Glu Gln LeuArg Gln Glu Pro Trp Tyr His Gly Arg Met Ser Arg 1 5 10 15 Arg Ala AlaGlu Arg Met Leu Arg Ala Asp Gly Asp Phe Leu Val 20 25 30 Arg Asp Ser ValThr Asn Pro Gly Gln Tyr Val Gly Met His Ala 35 40 45 Gly Gln Pro Lys HisLeu Leu Leu Val Asp Pro Glu Gly Val Val 50 55 60 Arg Thr Lys Asp Val LeuPhe Glu Ser Ile Ser His Leu Ile Asp 65 70 75 His His Leu Gln Asn Pro IleVal Ala Ala Glu Ser Glu Leu His 80 85 90 Leu Arg Gly Val Val Ser Arg GluPro 95 99 18 115 PRT Homo sapiens 18 Lys Pro Leu His Glu Gln Leu Trp TyrHis Gly Ala Ile Pro Arg 1 5 10 15 Ala Glu Val Ala Glu Leu Leu Val HisSer Gly Asp Phe Leu Val 20 25 30 Arg Glu Ser Gln Gly Lys Gln Glu Tyr ValVal Leu Trp Asp Gly 35 40 45 Leu Pro Arg His Phe Ile Ile Gln Ser Leu AspAsn Leu Tyr Arg 50 55 60 Leu Glu Gly Glu Gly Phe Pro Ser Ile Pro Leu LeuIle Asp His 65 70 75 Leu Leu Ser Thr Pro Leu Thr Lys Lys Ser Gly Val ValLeu His 80 85 90 Arg Ala Val Pro Lys Asp Lys Trp Val Leu Asn His Glu AspLeu 95 100 105 Val Leu Gly Glu Gln Ile Gly Arg Gly Asn 110 115 19 8 PRTHomo sapiens 19 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 8 20 688 PRT HomoSapiens 20 Ala Ala Gly Glu Pro Glu Ala Gly Ser Asp Tyr Val Lys Phe Ser 15 10 15 Lys Glu Lys Tyr Ile Leu Asp Ser Ser Pro Glu Lys Leu His Lys 2025 30 Glu Leu Glu Glu Glu Leu Lys Leu Ser Ser Thr Asp Leu Arg Ser 35 4045 His Ala Trp Tyr His Gly Arg Ile Pro Arg Glu Val Ser Glu Thr 50 55 60Leu Val Gln Arg Asn Gly Asp Phe Leu Ile Arg Asp Ser Leu Thr 65 70 75 SerLeu Gly Asp Tyr Val Leu Thr Cys Arg Trp Arg Asn Gln Ala 80 85 90 Leu HisPhe Lys Ile Asn Lys Val Val Val Lys Ala Gly Glu Ser 95 100 105 Tyr ThrHis Ile Gln Tyr Leu Phe Glu Gln Glu Ser Phe Asp His 110 115 120 Val ProAla Leu Val Arg Tyr His Val Gly Ser Arg Lys Ala Val 125 130 135 Ser GluGln Ser Gly Ala Ile Ile Tyr Cys Pro Val Asn Arg Thr 140 145 150 Phe ProLeu Arg Tyr Leu Glu Ala Ser Tyr Gly Leu Gly Gln Gly 155 160 165 Ser SerLys Pro Ala Ser Pro Val Ser Pro Ser Gly Pro Lys Gly 170 175 180 Ser HisMet Lys Arg Arg Ser Val Thr Met Thr Asp Gly Leu Thr 185 190 195 Ala AspLys Val Thr Arg Ser Asp Gly Cys Pro Thr Ser Thr Ser 200 205 210 Leu ProArg Pro Arg Asp Ser Ile Arg Ser Cys Ala Leu Ser Met 215 220 225 Asp GlnIle Pro Asp Leu His Ser Pro Met Ser Pro Ile Ser Glu 230 235 240 Ser ProSer Ser Pro Ala Tyr Ser Thr Val Thr Arg Val His Ala 245 250 255 Ala ProAla Ala Pro Ser Ala Thr Ala Leu Pro Ala Ser Pro Val 260 265 270 Ala ArgArg Ser Ser Glu Pro Gln Leu Cys Pro Gly Ser Ala Pro 275 280 285 Lys ThrHis Gly Glu Ser Asp Lys Gly Pro His Thr Ser Pro Ser 290 295 300 His ThrLeu Gly Lys Ala Ser Pro Ser Pro Ser Leu Ser Ser Tyr 305 310 315 Ser AspPro Asp Ser Gly His Tyr Cys Gln Leu Gln Pro Pro Val 320 325 330 Arg GlySer Arg Glu Trp Ala Ala Thr Glu Thr Ser Ser Gln Gln 335 340 345 Ala ArgSer Tyr Gly Glu Arg Leu Lys Glu Leu Ser Glu Asn Gly 350 355 360 Ala ProGlu Gly Asp Trp Gly Lys Thr Phe Thr Val Pro Ile Val 365 370 375 Glu ValThr Ser Ser Phe Asn Pro Ala Thr Phe Gln Ser Leu Leu 380 385 390 Ile ProArg Asp Asn Arg Pro Leu Glu Val Gly Leu Leu Arg Lys 395 400 405 Val LysGlu Leu Leu Ala Glu Val Asp Ala Arg Thr Leu Ala Arg 410 415 420 His ValThr Lys Val Asp Cys Leu Val Ala Arg Ile Leu Gly Val 425 430 435 Thr LysGlu Met Gln Thr Leu Met Gly Val Arg Trp Gly Met Glu 440 445 450 Leu LeuThr Leu Pro His Gly Arg Gln Leu Arg Leu Asp Leu Leu 455 460 465 Glu ArgPhe His Thr Met Ser Ile Met Leu Ala Val Asp Ile Leu 470 475 480 Gly CysThr Gly Ser Ala Glu Glu Arg Ala Ala Leu Leu His Lys 485 490 495 Thr IleGln Leu Ala Ala Glu Leu Arg Gly Thr Met Gly Asn Met 500 505 510 Phe SerPhe Ala Ala Val Met Gly Ala Leu Asp Met Ala Gln Ile 515 520 525 Ser ArgLeu Glu Gln Thr Trp Val Thr Leu Arg Gln Arg His Thr 530 535 540 Glu GlyAla Ile Leu Tyr Glu Lys Lys Leu Lys Pro Phe Leu Lys 545 550 555 Ser LeuAsn Glu Gly Lys Glu Gly Pro Pro Leu Ser Asn Thr Thr 560 565 570 Phe ProHis Val Leu Pro Leu Ile Thr Leu Leu Glu Cys Asp Ser 575 580 585 Ala ProPro Glu Gly Pro Glu Pro Trp Gly Ser Thr Glu His Gly 590 595 600 Val GluVal Val Leu Ala His Leu Glu Ala Ala Arg Thr Val Ala 605 610 615 His HisGly Gly Leu Tyr His Thr Asn Ala Glu Val Lys Leu Gln 620 625 630 Gly PheGln Ala Arg Pro Glu Leu Leu Glu Val Phe Ser Thr Glu 635 640 645 Phe GlnMet Arg Leu Leu Trp Gly Ser Gln Gly Ala Ser Ser Ser 650 655 660 Gln AlaArg Arg Tyr Glu Lys Phe Asp Lys Val Leu Thr Ala Leu 665 670 675 Ser HisLys Leu Glu Pro Ala Val Arg Ser Ser Glu Leu 680 685 688 21 576 PRTArtificial Sequence Artificial Sequence 1-576 21 Met Gln Val Pro Gln AspGly Glu Asp Leu Ala Gly Gln Pro Trp 1 5 10 15 Phe His Gly Leu Leu SerArg Gln Lys Ala Glu Ala Leu Leu Gln 20 25 30 Gln Asp Gly Asp Phe Leu ValArg Ala Ser Gly Ser Arg Gly Gly 35 40 45 Asn Pro Val Ile Ser Cys Arg TrpArg Gly Ser Ala Leu His Phe 50 55 60 Glu Val Phe Arg Val Ala Leu Arg ProArg Pro Gly Arg Pro Thr 65 70 75 Ala Leu Phe Gln Leu Glu Asp Glu Gln PhePro Ser Ile Pro Ala 80 85 90 Leu Val His Ser Tyr Met Thr Gly Arg Arg ProLeu Ser Gln Ala 95 100 105 Thr Gly Ala Val Val Ser Arg Pro Val Thr TrpGln Gly Pro Leu 110 115 120 Arg Arg Ser Phe Ser Glu Asp Thr Leu Met AspGly Pro Ala Arg 125 130 135 Ile Glu Pro Leu Arg Ala Arg Lys Trp Ser AsnSer Gln Pro Ala 140 145 150 Asp Leu Ala His Met Gly Arg Ser Arg Glu AspPro Ala Gly Met 155 160 165 Glu Ala Ser Thr Met Pro Ile Ser Ala Leu ProArg Thr Ser Ser 170 175 180 Asp Pro Val Leu Leu Lys Ala Pro Ala Pro LeuGly Thr Val Ala 185 190 195 Asp Ser Leu Arg Ala Ser Asp Gly Gln Leu GlnAla Lys Ala Pro 200 205 210 Thr Lys Pro Pro Arg Thr Pro Ser Phe Glu LeuPro Asp Ala Ser 215 220 225 Glu Arg Pro Pro Thr Tyr Cys Glu Leu Val ProArg Val Pro Ser 230 235 240 Val Gln Gly Thr Ser Pro Ser Gln Ser Cys ProGlu Pro Glu Ala 245 250 255 Pro Trp Trp Glu Ala Glu Glu Asp Glu Glu GluGlu Asn Arg Cys 260 265 270 Phe Thr Arg Pro Gln Ala Glu Ile Ser Phe CysPro His Asp Ala 275 280 285 Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg ProLeu Glu Pro Gln 290 295 300 Val Leu His Thr Leu Arg Gly Leu Phe Leu GluHis His Pro Gly 305 310 315 Ser Thr Ala Leu His Leu Leu Leu Val Asp CysGln Ala Thr Gly 320 325 330 Leu Leu Gly Val Thr Arg Asp Gln Arg Gly AsnMet Gly Val Ser 335 340 345 Ser Gly Leu Glu Leu Leu Thr Leu Pro His GlyHis His Leu Arg 350 355 360 Leu Glu Leu Leu Glu Arg His Gln Thr Leu AlaLeu Ala Gly Ala 365 370 375 Leu Ala Val Leu Gly Cys Ser Gly Pro Leu GluGlu Arg Ala Ala 380 385 390 Ala Leu Arg Gly Leu Val Glu Leu Ala Leu AlaLeu Arg Pro Gly 395 400 405 Ala Ala Gly Asp Leu Pro Gly Leu Ala Ala ValMet Gly Ala Leu 410 415 420 Leu Met Pro Gln Val Ser Arg Leu Glu His ThrTrp Arg Gln Leu 425 430 435 Arg Arg Ser His Thr Glu Ala Ala Leu Ala PheGlu Gln Glu Leu 440 445 450 Lys Pro Leu Met Arg Ala Leu Asp Glu Gly AlaGly Pro Cys Asp 455 460 465 Pro Gly Glu Val Ala Leu Pro His Val Ala ProMet Val Arg Leu 470 475 480 Leu Glu Gly Glu Glu Val Ala Gly Pro Leu AspGlu Ser Cys Glu 485 490 495 Arg Leu Leu Arg Thr Leu His Gly Ala Arg HisMet Val Arg Asp 500 505 510 Ala Pro Lys Phe Arg Lys Val Ala Ala Gln ArgLeu Arg Gly Phe 515 520 525 Arg Pro Asn Pro Glu Leu Arg Glu Ala Leu ThrThr Gly Phe Val 530 535 540 Arg Arg Leu Leu Trp Gly Ser Arg Gly Ala GlyAla Pro Arg Ala 545 550 555 Glu Arg Phe Glu Lys Phe Gln Arg Val Leu GlyVal Leu Ser Gln 560 565 570 Arg Leu Glu Pro Asp Arg 575 576 22 576 PRTArtificial Sequence Artificial Sequence 1-576 22 Met Gln Val Pro Gln AspGly Glu Asp Leu Ala Gly Gln Pro Trp 1 5 10 15 Tyr His Gly Leu Leu SerArg Gln Lys Ala Glu Ala Leu Leu Gln 20 25 30 Gln Asp Gly Asp Phe Leu ValArg Ala Ser Gly Ser Arg Gly Gly 35 40 45 Asn Pro Val Ile Ser Cys Arg TrpArg Gly Ser Ala Leu His Phe 50 55 60 Glu Val Phe Arg Val Ala Leu Arg ProArg Pro Gly Arg Pro Thr 65 70 75 Ala Leu Phe Gln Leu Glu Asp Glu Gln PhePro Ser Ile Pro Ala 80 85 90 Leu Val His Ser Phe Met Thr Gly Arg Arg ProLeu Ser Gln Ala 95 100 105 Thr Gly Ala Val Val Ser Arg Pro Val Thr TrpGln Gly Pro Leu 110 115 120 Arg Arg Ser Phe Ser Glu Asp Thr Leu Met AspGly Pro Ala Arg 125 130 135 Ile Glu Pro Leu Arg Ala Arg Lys Trp Ser AsnSer Gln Pro Ala 140 145 150 Asp Leu Ala His Met Gly Arg Ser Arg Glu AspPro Ala Gly Met 155 160 165 Glu Ala Ser Thr Met Pro Ile Ser Ala Leu ProArg Thr Ser Ser 170 175 180 Asp Pro Val Leu Leu Lys Ala Pro Ala Pro LeuGly Thr Val Ala 185 190 195 Asp Ser Leu Arg Ala Ser Asp Gly Gln Leu GlnAla Lys Ala Pro 200 205 210 Thr Lys Pro Pro Arg Thr Pro Ser Phe Glu LeuPro Asp Ala Ser 215 220 225 Glu Arg Pro Pro Thr Tyr Cys Glu Leu Val ProArg Val Pro Ser 230 235 240 Val Gln Gly Thr Ser Pro Ser Gln Ser Cys ProGlu Pro Glu Ala 245 250 255 Pro Trp Trp Glu Ala Glu Glu Asp Glu Glu GluGlu Asn Arg Cys 260 265 270 Phe Thr Arg Pro Gln Ala Glu Ile Ser Phe CysPro His Asp Ala 275 280 285 Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg ProLeu Glu Pro Gln 290 295 300 Val Leu His Thr Leu Arg Gly Leu Phe Leu GluHis His Pro Gly 305 310 315 Ser Thr Ala Leu His Leu Leu Leu Val Asp CysGln Ala Thr Gly 320 325 330 Leu Leu Gly Val Thr Arg Asp Gln Arg Gly AsnMet Gly Val Ser 335 340 345 Ser Gly Leu Glu Leu Leu Thr Leu Pro His GlyHis His Leu Arg 350 355 360 Leu Glu Leu Leu Glu Arg His Gln Thr Leu AlaLeu Ala Gly Ala 365 370 375 Leu Ala Val Leu Gly Cys Ser Gly Pro Leu GluGlu Arg Ala Ala 380 385 390 Ala Leu Arg Gly Leu Val Glu Leu Ala Leu AlaLeu Arg Pro Gly 395 400 405 Ala Ala Gly Asp Leu Pro Gly Leu Ala Ala ValMet Gly Ala Leu 410 415 420 Leu Met Pro Gln Val Ser Arg Leu Glu His ThrTrp Arg Gln Leu 425 430 435 Arg Arg Ser His Thr Glu Ala Ala Leu Ala PheGlu Gln Glu Leu 440 445 450 Lys Pro Leu Met Arg Ala Leu Asp Glu Gly AlaGly Pro Cys Asp 455 460 465 Pro Gly Glu Val Ala Leu Pro His Val Ala ProMet Val Arg Leu 470 475 480 Leu Glu Gly Glu Glu Val Ala Gly Pro Leu AspGlu Ser Cys Glu 485 490 495 Arg Leu Leu Arg Thr Leu His Gly Ala Arg HisMet Val Arg Asp 500 505 510 Ala Pro Lys Phe Arg Lys Val Ala Ala Gln ArgLeu Arg Gly Phe 515 520 525 Arg Pro Asn Pro Glu Leu Arg Glu Ala Leu ThrThr Gly Phe Val 530 535 540 Arg Arg Leu Leu Trp Gly Ser Arg Gly Ala GlyAla Pro Arg Ala 545 550 555 Glu Arg Phe Glu Lys Phe Gln Arg Val Leu GlyVal Leu Ser Gln 560 565 570 Arg Leu Glu Pro Asp Arg 575 576 23 576 PRTArtificial Sequence Artificial Sequence 1-576 23 Met Gln Val Pro Gln AspGly Glu Asp Leu Ala Gly Gln Pro Trp 1 5 10 15 Tyr His Gly Leu Leu SerArg Gln Lys Ala Glu Ala Leu Leu Gln 20 25 30 Gln Asp Gly Asp Phe Leu ValArg Ala Ser Gly Ser Arg Gly Gly 35 40 45 Asn Pro Val Ile Ser Cys Arg TrpArg Gly Ser Ala Leu His Phe 50 55 60 Glu Val Phe Arg Val Ala Leu Arg ProArg Pro Gly Arg Pro Thr 65 70 75 Ala Leu Phe Gln Leu Glu Asp Glu Gln PhePro Ser Ile Pro Ala 80 85 90 Leu Val His Ser Tyr Met Thr Gly Arg Arg ProLeu Ser Gln Ala 95 100 105 Thr Gly Ala Val Val Ser Arg Pro Val Thr TrpGln Gly Pro Leu 110 115 120 Arg Arg Ser Phe Ser Glu Asp Thr Leu Met AspGly Pro Ala Arg 125 130 135 Ile Glu Pro Leu Arg Ala Arg Lys Trp Ser AsnSer Gln Pro Ala 140 145 150 Asp Leu Ala His Met Gly Arg Ser Arg Glu AspPro Ala Gly Met 155 160 165 Glu Ala Ser Thr Met Pro Ile Ser Ala Leu ProArg Thr Ser Ser 170 175 180 Asp Pro Val Leu Leu Lys Ala Pro Ala Pro LeuGly Thr Val Ala 185 190 195 Asp Ser Leu Arg Ala Ser Asp Gly Gln Leu GlnAla Lys Ala Pro 200 205 210 Thr Lys Pro Pro Arg Thr Pro Ser Phe Glu LeuPro Asp Ala Ser 215 220 225 Glu Arg Pro Pro Thr Phe Cys Glu Leu Val ProArg Val Pro Ser 230 235 240 Val Gln Gly Thr Ser Pro Ser Gln Ser Cys ProGlu Pro Glu Ala 245 250 255 Pro Trp Trp Glu Ala Glu Glu Asp Glu Glu GluGlu Asn Arg Cys 260 265 270 Phe Thr Arg Pro Gln Ala Glu Ile Ser Phe CysPro His Asp Ala 275 280 285 Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg ProLeu Glu Pro Gln 290 295 300 Val Leu His Thr Leu Arg Gly Leu Phe Leu GluHis His Pro Gly 305 310 315 Ser Thr Ala Leu His Leu Leu Leu Val Asp CysGln Ala Thr Gly 320 325 330 Leu Leu Gly Val Thr Arg Asp Gln Arg Gly AsnMet Gly Val Ser 335 340 345 Ser Gly Leu Glu Leu Leu Thr Leu Pro His GlyHis His Leu Arg 350 355 360 Leu Glu Leu Leu Glu Arg His Gln Thr Leu AlaLeu Ala Gly Ala 365 370 375 Leu Ala Val Leu Gly Cys Ser Gly Pro Leu GluGlu Arg Ala Ala 380 385 390 Ala Leu Arg Gly Leu Val Glu Leu Ala Leu AlaLeu Arg Pro Gly 395 400 405 Ala Ala Gly Asp Leu Pro Gly Leu Ala Ala ValMet Gly Ala Leu 410 415 420 Leu Met Pro Gln Val Ser Arg Leu Glu His ThrTrp Arg Gln Leu 425 430 435 Arg Arg Ser His Thr Glu Ala Ala Leu Ala PheGlu Gln Glu Leu 440 445 450 Lys Pro Leu Met Arg Ala Leu Asp Glu Gly AlaGly Pro Cys Asp 455 460 465 Pro Gly Glu Val Ala Leu Pro His Val Ala ProMet Val Arg Leu 470 475 480 Leu Glu Gly Glu Glu Val Ala Gly Pro Leu AspGlu Ser Cys Glu 485 490 495 Arg Leu Leu Arg Thr Leu His Gly Ala Arg HisMet Val Arg Asp 500 505 510 Ala Pro Lys Phe Arg Lys Val Ala Ala Gln ArgLeu Arg Gly Phe 515 520 525 Arg Pro Asn Pro Glu Leu Arg Glu Ala Leu ThrThr Gly Phe Val 530 535 540 Arg Arg Leu Leu Trp Gly Ser Arg Gly Ala GlyAla Pro Arg Ala 545 550 555 Glu Arg Phe Glu Lys Phe Gln Arg Val Leu GlyVal Leu Ser Gln 560 565 570 Arg Leu Glu Pro Asp Arg 575 576 24 20 DNAArtificial Sequence Artificial Sequence 1-20 24 cgcagacacc cttcttcaca 2025 22 DNA Artificial Sequence Artificial Sequence 1-22 25 cgactcctttggtctcttct gg 22 26 21 DNA Artificial Sequence Artificial Sequence 1-2126 ccgggacccc caggtttttg c 21 27 19 DNA Artificial Sequence ArtificialSequence 1-19 27 agggtcctgc gtggactct 19 28 23 DNA Artificial SequenceArtificial Sequence 1-23 28 tcctgttctt cctcaatgga gac 23 29 25 DNAArtificial Sequence Artificial Sequence 1-25 29 ccatcccacc tgctacatgctcacc 25 30 48 DNA Artificial Sequence Artificial Sequence 1-48 30ggattctaat acgactcact atagggcgcg gaggctgctc tggggtag 48 31 48 DNAArtificial Sequence Artificial Sequence 1-48 31 ctatgaaatt aaccctcactaaagggatgt tgccctggct ggtcttga 48 32 48 DNA Artificial SequenceArtificial Sequence 1-48 32 ggattctaat acgactcact atagggcatc tgccttgccccgaacgag 48 33 48 DNA Artificial Sequence Artificial Sequence 1-48 33ctatgaaatt aaccctcact aaagggatca tccagagccc gcatcagc 48 34 48 DNAArtificial Sequence Artificial Sequence 1-48 34 ggattctaat acgactcactatagggcaga tgtggaagac tgaggcct 48 35 49 DNA Artificial SequenceArtificial Sequence 35 ctatgaaatt aaccctcact aaagggaata tgtgccaaatctgcaggct 49

What is claimed is:
 1. Isolated nucleic acid comprising DNA having atleast a 90% sequence identity to (a) a DNA molecule encoding a PRO201polypeptide comprising the sequence of amino acids 1 to 576 of FIG. 1(SEQ ID NO: 1), or (b) the complement of the DNA molecule of (a).
 2. Theisolated nucleic acid comprising DNA having at least a 90% sequenceidentity to (a) a DNA molecule encoding a PRO309 polypeptide comprisingthe sequence of amino acids 1 to 703 of FIG. 3 (SEQ ID NO: 5), or (b)the complement of the DNA molecule of (a).
 3. The isolated nucleic acidof claim 1 comprising DNA encoding a PRO201 polypeptide having aminoacid residues 1 to 576 of FIG. 1 (SEQ ID NO: 1).
 4. The isolated nucleicacid of claim 2 comprising DNA encoding a PRO309 polypeptide havingamino acid residues 1 to 703 of FIG. 3 (SEQ ID NO: 5).
 5. An isolatednucleic acid comprising DNA having at least a 95% sequence identity to(a) a DNA molecule encoding the same mature polypeptide encoded by thecDNA in ATCC Deposit No. 209567 (designation: DNA30676-1223), or (b) thecomplement of the DNA molecule of (a).
 6. An isolated nucleic acidcomprising DNA having at least a 95% sequence identity to (a) a DNAmolecule encoding the same mature polypeptide encoded by the cDNA inATCC Deposit No. 209713, or (b) the complement of the DNA molecule of(a).
 7. A vector comprising the nucleic acid of claim
 1. 8. A vectorcomprising the nucleic acid of claim
 2. 9. The vector of claim 7operably linked to control sequences recognized by a host celltransformed with the vector.
 10. The vector of claim 8 operably linkedto control sequences recognized by a host cell transformed with thevector.
 11. A host cell comprising the vector of claim
 9. 12. A hostcell comprising the vector of claim
 10. 13. The host cell of claim 11wherein said cell is mammalian.
 14. The host cell of claim 13 whereinsaid cell is a CHO cell.
 15. The host cell of claim 11 wherein said cellis prokaryotic.
 16. The host cell of claim 15 wherein said cell is an E.coli.
 17. The host cell of claim 11 wherein said cell is a yeast cell.18. The host cell of claim 17 wherein said cell is Saccharomycescerevisiae.
 19. A process for producing PRO201, PRO308 or PRO309polypeptides comprising culturing the host cell of claim 6 underconditions suitable for expression of PRO201, PRO308 or PRO309,respectively, and recovering PRO201, PRO308 or PRO309, respectively,from the cell culture.
 20. Isolated native sequence PRO201 polypeptidecomprising amino acid residues 1 to 576 of FIG. 1 (SEQ ID NO: 1). 21.Isolated native sequence PRO309 polypeptide comprising amino acidresidues 1 to 703 of FIG. 3 (SEQ ID NO: 5).
 22. Isolated native sequencePRO201 polypeptide encoded by the nucleotide deposited under accessionnumber ATCC
 209567. 23. Isolated native sequence PRO309 polypeptideencoded by the nucleotide deposited under accession number ATCC 209713.24. A chimeric molecule comprising PRO201, PRO308 or PRO309 polypeptidefused to a heterologous amino acid sequence.
 25. The chimeric moleculeof claim 24 wherein said heterologous amino acid sequence is an epitopetag sequence.
 26. The chimeric molecule of claim 25 wherein saidheterologous amino acid sequence is a Fc region of an immunoglobulin.27. An antagonist of Nsp1 (SEQ ID NO:1), Nsp2 (SEQ ID NO:3) or Nsp3 (SEQID NO:5) which blocks, prevents, inhibits and/or otherwise neutralizesthe normal functioning of Nsp1 (SEQ ID NO:1), Nsp2 (SEQ ID NO:3) or Nsp3(SEQ ID NO:5), respectively, in cellular signaling.
 28. The antagonistof claim 27 which is a small bioorganic molecule.
 29. The antagonist ofclaim 28 which is an antisense oligonucleotide.
 30. An antibody whichspecifically binds to PRO201, PRO308 or PRO309 polypeptide.
 31. Theantibody of claim 30 wherein said antibody is a monoclonal antibody. 32.The antibody of claim 31 which induces the death of a celloverexpressing PRO201, PRO308 or PRO309 polypeptide.
 33. The antibody ofclaim 32 wherein said cell is a tumor cell.
 34. The antibody of claim 31which is labeled.
 35. The antibody of claim 34 which is immobilized on asolid support.
 36. The antibody of claim 34 which is an active fragment,single-chain antibody or an anti-idiotypic antibody.
 37. A compositioncomprising an antibody of claim 30 in admixture with a pharmaceuticallyacceptable carrier.
 38. The composition of claim 37 comprising a growthinhibitory amount of said antibody.
 39. The composition of claim 38further comprising a second antibody or a cytotoxic or chemotherapeuticagent.
 40. A method for determining the presence of a PRO201, PRO308 orPRO309 polypeptide comprising exposing a cell suspected of containingPRO201, PRO308 or PRO309 or expressing DNA encoding said polypeptide toanti-PRO201, anti-PRO308 or anti-PRO309 and determining the binding ofsaid antibody to said cell.
 41. A method of diagnosing tumor in amammal, comprising detecting the level of expression of a gene encodinga PRO201, PRO308 or PRO309 polypeptide (a) in a test sample of tissuecells obtained from the mammal, and (b) in a control sample of knownnormal tissue cells of the same cell type, wherein a higher expressionlevel in the test sample indicated the presence of tumor in the mammalfrom which the test tissue cells were obtained.
 42. A method ofdiagnosing tumor in a mammal, comprising (a) contacting a anti-PRO201,anti-PRO308 or anti-PRO309 antibody with a test sample of tissue cellsobtained from the mammal, and (b) detecting the formation of a complexbetween the anti-PRO201, anti-PRO308 or anti-PRO309 antibody and thePRO201, PRO308 or PRO309 polypeptide in the test sample.
 43. The methodof claim 42 wherein said test sample is obtained from a mammal suspectedto have neoplastic cell growth or proliferation.
 44. A cancer diagnostickit, comprising an anti-PRO201, anti-PRO308 or anti-PRO309 antibody anda carrier in suitable packaging.
 45. A method for inhibiting the growthof tumor cells comprising exposing a cell which overexpresses a PRO201,PRO308 or PRO309 polypeptide to an effective amount of an agentinhibiting the expression and/or activity of the PRO201, PRO308 orPRO309 polypeptide.
 46. The method of claim 45, wherein said agent is ananti-PRO201, anti-PRO308 or anti-PRO309 antibody.
 47. The method ofclaim 46 wherein said tumor cells are further exposed to radiationtreatment or a cytotoxic or chemotherapeutic agent.